Primerdesign Catalogue / Technical Advice 2019

Products for Real-Time PCR by Primerdesign

Contents 38

oasig® PLEX Lyophilised OneStep RT-qPCR Master Mix

genesig® Easy Magnetic Rack

40

BrightWhite Real-Time PCR qPCR Plates

8

Precision™ DNase Kits

42

genesig® Easy Tips

10

Precision™ Reverse Transcription Kits

43

RNase/DNase Free Water

12

Precision™ Reverse Transcription Premix

44

BioBank Control cDNA

14

geNorm™ Reference Gene Selection Kits

46

Real-Time PCR Internal Control Kit

16

geNormPLUS Reference Gene Selection Kits

48

Positive Control Template for Standard Curve

18

qbase+

50

Genomic DNA Detection Kits

20

Reference Gene Assays

51

Y Chromosome Detection Kits

22

Custom Gene Of Interest Real-Time PCR Assays

52

24

genesig® q16 and genesig® q32

Molecular Diagnostics (MDx) Assay Development Services

26

genesig® Kits

54

Technical Bulletins

28

Precision®PLUS qPCR Master Mix

82

The MIQE Guidelines

30

Precision®PLUS OneStep RT-qPCR Master Mix

32

Precision®FAST qPCR Master Mix

34

oasig®Lyophilised 2X qPCR and oasig® Lyophilised 2X OneStep RT-qPCR Master Mix

36

oasig® PLEX Lyophilised qPCR Master Mix

3

Introduction

4

genesig® Easy DNA/RNA Extraction Kit

6

2

87 Appendix

Introduction Novacyt is a rapidly growing, international diagnostics group, generating revenues from the sale of clinical products used in oncology, microbiology, haematology and serology testing.

The Group has considerable experience in the development, manufacture and commercialisation of molecular, protein and whole-cell diagnostic products and aims to become a leader in developing new products for the infectious disease and oncology testing markets. The Group has a strong intellectual property portfolio and considerable product and process ‘know-how’ in the key technologies used across its operating segments.

Primerdesign, part of the Novacyt Group, was formed in 2004 to share the expertise in real-time PCR and DNA chemistry found within the University of Southampton. Since then we have grown substantially, developing thousands of products, helping thousands of customers and working with customers in 100+ countries around the World.

Our core expertise is in the development of real-time PCR assays.

We can produce real-time PCR assays for any gene of interest in any species. Our kits are available in a range of formats but, whatever your preference, you can be sure that every set of primers is designed by an expert, synthesised, and then fully validated on real biological cDNA in our laboratories to ensure that it is guaranteed to work to the highest standards on the day the kit arrives with you.

In addition to the World’s best custom designed primers, we can also provide everything else you need to perform real-time PCR to the highest standards whilst keeping your costs under control.

And of course, our dedicated, friendly team of experts are available to support you technically throughout your project to help you achieve the highest quality of data for your next publication or project.

Uniquely, we will also provide all of the primer details for any OnDemand Kits that we produce for you to give you complete scientific integrity in your research. This makes our kits compliant with the The MIQE Guidelines: Minimum Information for Publication of Quantitative real-time PCR Experiments; Bustin S.A., et al 2009.

Clinical Chemistry 55(4) 611–622

3

genesig® Easy DNA/RNA Extraction Kit Universal kit for isolation of DNA/RNA from food, water, clinical, veterinary and other samples types.

The genesig Easy DNA/RNA Extraction Kit allows DNA and RNA extraction from virtually any sample type using magnetic bead technology - it’s fast, and incredibly easy to perform.

Product features • Extracts DNA and RNA with high yields in 60 minutes • Works with huge range of sample types • Safe protocol with no phenolic chemicals • No centrifuge required - for use in the lab or in the field

The genesig Easy DNA/RNA extraction protocol begins with a simple lysis step where cells and tissue are lysed to release their nucleic acid. Then tiny magnetic particles are added to bind to DNA/RNA. When placed on to the genesig magnetic separator the particles are pulled to the side of the tube making it easy to remove the unwanted supernatant with a pipette. Then a series of simple wash steps are performed before the DNA/RNA is washed off the beads back in to solution, ready for analysis by real-time PCR. Suitable sample types: • Clinical and Veterinary - including whole blood, plasma, and serum; saliva and sputum; faeces and urine; tissues; and bacterial culture broth and swabs • Food - including meat, fish, alcohol, milk, and cooked or processed food products • Environmental - including plant, soil, and water

4

DNA & RNA extraction from virtually any sample

GENESIG® EASY DNA/RNA EXTRACTION KITS CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-genesigEASY-EK

genesig Easy DNA/RNA Extraction Kit

50 extractions

5

genesig® Easy Magnetic Rack The genesig Easy Magnetic Rack for use with the genesig Easy DNA/ RNA Extraction Kit enables simple nucleic acid extraction from up to 16 different samples at once. Powerful magnets and angled sides create very tight magnetic bead clumps. This makes it simple to aspirate from the tube with a pipette without the risk of disturbing your magnetic beads.

The detachable tube holder makes it convenient to handle multiple samples simultaneously. When used in combination with the genesig Easy DNA/RNA Extraction Kit, nucleic acid extraction from virtually any sample is made easy.

Product features • For use with genesig Easy DNA/RNA Extraction Kit • Powerful magnets for tight magnetic bead clumps and easy aspiration • Extraction from up to 16 different samples at once

6

GENESIG® EASY MAGNETIC RACK CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-genesigEASY-MR

genesig Easy Magnetic Rack for DNA/RNA extraction

1 rack for 16 samples

Extraction from up to 16 different samples at once

7

Precision™ DNase Kits Precision DNase is a novel product designed specifically for removing gDNA contamination from RNA prior to reverse transcription/real-time PCR. The Precision DNase enzyme has an exceptional level of specificity towards dsDNA and no activity against RNA. In addition the kit’s novel deactivation mechanism results in pure, un-degraded RNA. Unique ‘stop’ method protects RNA; our enzyme is inactivated by a short incubation at just 55°C. Heat inactivation above 65°C, as common for other enzymes, can cause magnesium mediated degradation of RNA. This simple inactivation method ensures that RNA is both protected from degradation and prevents the need for additional reagents to stop the reaction that may affect sample quality.

Other DNase kits available are unsuitable for preparing RNA for realtime PCR. • Many use an aggressive heat inactivation (>65°C) which can result in magnesium mediated RNA degradation • Some use a chemical stop solution containing EDTA (a known disrupter of PCR) • Whilst other kits offer a slurry of beads that capture the enzyme and allow centrifugal removal. This final approach makes recovering all of the RNA sample impossible Our Precision DNase kit is unique in having a heat labile enzyme.

This means that enzyme activity is entirely destroyed by an ‘RNA safe’ 5 minute incubation at 65°C.

8

Product features • Precise and efficient gDNA removal in 15 minutes • No damage to RNA • No addition of EDTA (PCR inhibitor) • 100% recovery of all RNA This product has been designed and manufactured in collaboration with ArticZymes AS

PRECISION DNASE KIT CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-DNase-50

Precision DNase kit

50 reactions

Precise and efficient gDNA removal in 15 minutes 9

Precision™ Reverse Transcription Kits High efficiency reverse transcription (RT) ensures the ability to detect rare transcripts and obtain the strongest possible real-time PCR signals. As a complete solution, the Primerdesign Reverse Transcription kit contains all of the reagents necessary. Both Oligo(dT) and random nonamers are included for complete flexibility.

For advice on the most appropriate priming strategy contact our team of experts on support@primerdesign.co.uk Our reverse transcriptase are especially modified to work at high temperatures. Higher reaction temperatures reduce the RNA secondary structure (tangling), allow the production of longer transcripts and make for faster reactions. The enzyme also has an increased affinity for primer-template complexes which allow for efficient transcription of very low amounts of RNA.

Kit contents • High quality nanoScript2 RT enzyme • 10 x RT buffer • DTT • Oligo(dT) primers • Random nonamer primers • dNTPs • RNase/DNase-free water

Product features • Cost saving • Precise, reproducible results • Ultra fast protocol • Flexible kit design - Oligo(dT), random nonamer

10

Precise, reproducible results

PRECISION REVERSE TRANSCRIPTION KITS CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-RT-nanoScript2

nanoScript2 Reverse Transcription kit suitable for 2 nanograms to 2 micrograms of RNA template

50 reactions

11

Precision™ Reverse Transcription Premix Our all-in-one RT products provide the ultimate convenience for cDNA synthesis.

The pre-optimised mix allows for rapid reverse transcription in a single closed tube reaction. Our proprietary formulation contains an optimised blend of oligo(dT) and random nonamer primers, buffer, dNTPs, and RT enzyme. Simply add RNA to the mix, incubate at 55°C for 20 minutes and your perfect reverse transcription reaction is complete.

Product features • Ultimate convenience • Reduced contamination risks • Reduced handling errors • Ultra fast protocol

12

The ultimate convenience for cDNA synthesis

REVERSE TRANSCRIPTION PREMIX CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-RT-premix2

All-in-one Reverse Transcription mix. Single tube for manual dispensing.

50 reactions

Z-RT-noRT-ctrl

No RT control aliquot (complete mix lacking the RT enzyme)

50 reactions

13

geNorm™ Reference Gene Selection Kits For accurate gene quantification, it is essential to normalise realtime PCR data to a fixed reference; one that is not affected by your experimental conditions. There is increasing evidence that normalising to a single, randomly selected reference (housekeeping) gene introduces large and variable errors into the analysis. The geNorm kit is a system for selecting the best candidate reference genes for a given experimental scenario.

>12,000 papers have already cited the geNorm method.

To perform geNorm analysis, the user measures the expression in their samples of the panel of candidate reference genes in the kit. The geNorm software qbase+ provided with the kit ranks the reference genes in order of stability. The output graphs identify the best reference genes for that particular experiment and also the optimum number that should be included for accurate normalisation.

Our classic geNorm kit contains the most commonly used reference genes in the literature plus additional genes from a wide variety of cellular processes. A full list of the genes in each kit is available on our website.

MIQE guidelines compliant

Product features • Quick simple protocol • Identifies the best reference genes for any given experimental model • Improves accuracy, sensitivity and reproducibility of subsequent experiments • Qualify for 50% off future reference gene assay orders*

Kit contents • Lyophilised primers (and probe) for 6 or 12 reference genes (200 reactions each) • RNase/DNase free water • Access to the latest geNorm software in qbase+ • 50% off discount code for future reference gene assay orders* In addition to human, mouse and rat we have geNorm kits for many other research species including E.Coli, Drosophila, Horse, Xenopus, Sheep, Zebra Fish, Chicken, Pig, Rabbit.

See our website for the latest complete list. If you would like to use geNorm for a species that we do not currently support then please contact us as we can develop kits on demand.

In collaboration with

* See page 20

14

GENORM™ REFERENCE GENE SELECTION KITS HUMAN CATALOGUE NO.

PRODUCT DESCRIPTION

Z-ge-SY-6-hu Z-ge-SY-12-hu Z-ge-DD-6-hu Z-ge-DD-12-hu

6 gene human geNorm kit for use with SYBR Green 12 gene human geNorm™ kit for use with SYBR Green 6 gene human geNorm™ kit with Double-Dye hydrolysis probe 12 gene human geNorm™ kit with Double-Dye hydrolysis probe

6 x 200 reactions 12 x 200 reactions 6 x 200 reactions 12 x 200 reactions

CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-ge-SY-6-mo Z-ge-SY-12-mo Z-ge-DD-6-mo Z-ge-DD-12-mo

6 gene mouse geNorm kit for use with SYBR Green 12 gene mouse geNorm™ kit for use with SYBR Green 6 gene mouse geNorm™ kit with Double-Dye hydrolysis probe 12 gene mouse geNorm™ kit with Double-Dye hydrolysis probe

6 x 200 reactions 12 x 200 reactions 6 x 200 reactions 12 x 200 reactions

CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-ge-SY-6-ra Z-ge-SY-12-ra Z-ge-DD-6-ra Z-ge-DD-12-ra

6 gene rat geNorm™ kit for use with SYBR Green 12 gene rat geNorm™ kit for use with SYBR Green 6 gene rat geNorm™ kit with Double-Dye hydrolysis probe 12 gene rat geNorm™ kit with Double-Dye hydrolysis probe

6 x 200 reactions 12 x 200 reactions 6 x 200 reactions 12 x 200 reactions

™

KIT SIZE ®

MOUSE ™

RAT

Genome Biology, 2002, ‘Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes’ Vandesompele et al

15

geNormPLUS Reference Gene Selection Kits What is different about a geNormPLUS kit?

Kit contents

In collaboration with Nebion (www.nebion.com), using their Genevestigator® technology, we screened data from >30,000 microarray experiments to identify genes that express inherent levels of stability across different tissues, alternative experimental treatments, different disease states and many other conditions. The result is a list of genes that are inherently stably expressed (the lists includes a selection of classically used reference (housekeeping) genes for comparison too). geNorm analysis with these genes therefore enables selection of the “best of the best”.

• Lyophilised primers for 6 or 12 reference genes (200 reactions each) • RNase/DNase free water • Access to the latest geNorm software in qbase+

A complete list of the genes included can be found on our website.

Product features • Scientifically selected, inherently stable genes • Quick simple protocol • Identifies the best reference genes for any given experimental model • Improves accuracy, sensitivity and reproducibility of subsequent experiments In collaboration with

16

GENORMPLUS REFERENCE GENE SELECTION KITS HUMAN CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-gePLUS-SY-6-hu Z-gePLUS -SY-12-hu

6 gene human geNormPLUS kit for use with SYBR Green 12 gene human geNormPLUS kit for use with SYBR Green

6 x 200 reactions 12 x 200 reactions

CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-gePLUS -SY-6-mo Z-gePLUS -SY-12-mo

6 gene mouse geNormPLUS kit for use with SYBR Green 12 gene mouse geNormPLUS kit for use with SYBR Green

6 x 200 reactions 12 x 200 reactions

CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-gePLUS -SY-6-ra Z-gePLUS -SY-12-ra

6 gene rat geNormPLUS kit for use with SYBR Green 12 gene rat geNormPLUS kit for use with SYBR Green

6 x 200 reactions 12 x 200 reactions

®

MOUSE

RAT

Scientifically selected, inherently stable genes 17

qbase+

the World’s best real-time PCR analysis software Primerdesign is delighted to have been chosen by Biogazelle to partner them in marketing their World famous qPCR analysis software qbase+. The software is recognised as the most powerful and user-friendly product in the marketplace. The software will completely automate analysis of your qPCR data whilst being flexible enough to produce the graphs and statistics that you need to tell your story of choice with your data.

Product features • Easy to use • Time saving • Flexible • Analysis wizard • Effortless scaling for larger experiments • Complete automation of statistics and graphing • Based on peer reviewed quantification models • MIQE and RDML ompliant In collaboration with

18

The most powerful and user-friendly software in the marketplace

QBASE+ CATALOGUE NO.

PRODUCT DESCRIPTION

LICENSES AVAILABLE

Z-qbase-acc-B

qbase licences for academic users

Premium 3 years Premium 1 year Premium 2 months Basic 1 year Basic 2 months

Z-qbase-com-B

qbase+ licences for commercial users

Premium 3 years Premium 1 year Premium 2 months Basic 1 year Basic 2 months

+

For group licenses please enquire.

19

Reference Gene Assays To compliment your custom gene of interest assay, Primerdesign has a wide range of reference gene assays which can be used as a normalising signal.

If you do not know the best reference genes for your experiment, you can use our geNorm kit to enable selection of the most stable genes in your individual experiment.

Please see Appendix on page 87 for a list of our available reference gene assays, or our website.

Wide range available! .

20

REFERENCE GENE ASSAYS HUMAN CATALOGUE NO. Z-HK-DD-hu-600

PRODUCT DESCRIPTION Pre-validated primers for your choice of human reference gene for use with SYBR® Green

KIT SIZE 600 reactions

PRODUCT DESCRIPTION Pre-validated primers for your choice of mouse reference gene for use with SYBR Green

KIT SIZE 600 reactions

PRODUCT DESCRIPTION Pre-validated primers for your choice of rat reference gene for use with SYBR Green

KIT SIZE 600 reactions

MOUSE CATALOGUE NO. Z-HK-DD-mo-600 RAT CATALOGUE NO. Z-HK-DD-ra-600

21

Custom Gene Of Interest Real-Time PCR Assays We offer specific real-time PCR assay development including primer/ probe design, PCR mix optimisation and assay development to ensure a very effective real-time PCR reaction.

We have already developed 1,000s of assays as requested from 100+ countries. This service allows for the combination of bioinformatic design and development, in conjunction with laboratory testing and evaluation, to provide personally designed assays to match your exact needs and ensure high levels of sensitivity and specificity.

Our core expertise is in the design of the very best real-time PCR assays. Every assay is individually designed to your requirements by an expert member of our team.

After synthesis it is then fully validated in our laboratory on relevant biologically derived gDNA/cDNA or synthetic target to ensure that the kit works to the highest standards.

What real-time PCR assay development do we offer: • Primer-only: This is typically the most basic form of design and can be used for standard as well as real-time PCR. As the name suggests, it is just a forward and reverse primer, which allows for dye-based chemistry such as SYBR®green.Designed by ‘in-house’ bioinformatical experts, not databases • Double Dye: This expands on primer-only assays with an inclusion of a double dye probe in the form of TaqMan® probe.

This is the most classical cleavage probe technology.

22

• Genotyping and SNP-Typing: Discrimination between genotypes is typically difficult and this is something we specialise in. Our unique in-house technology in combination with expert designers allows for a highly effective assay.

• Miscellaneous: Anything that doesn’t match the assay types above. We have expertise in so many niche and specialized realtime PCR technologies, so please get in touch and we’ll do our best to meet your requirements.

Upon request of development, one of our expert team members will be in contact to completely understand any specific requirements you may have (incl. exclusivity criteria, etc), and they are happy to talk you through the whole process.

Product features: • Top quality assays with guaranteed results • Designed by ‘in-house’ bioinformatical experts, not databases • Scientifically validated in our laboratory • An assay for almost any application • Improved genotyping discrimination

23

genesig® q16 and genesig® q32 extraordinary qPCR instruments and kits for real-time PCR genesig q16 Real-Time PCR Instrument

NEW! genesig q32 Real-Time PCR Instrument

Small and portable molecular testing platform

A fast qPCR instrument for all genesig kits

The genesig q16 Real-Time PCR Instrument is small and highly portable to allow any business to access the power of real-time PCR. Perfect for a whole new world of molecular testing customers .

The genesig q32 Real-Time PCR Instrument is one of the fastest qPCR instruments on the market today due to its rapid heating and cooling. Assembled from just a few building blocks, this robust qPCR instrument allows analysis of up to 32 samples in both tube or strip format.

Automated analysis The genesig q16 makes real-time PCR easy for the first time. The genesig Easy Kit format is designed specifically for the q16 and simplifies everything during test set-up. The q16 software experience is fast and intuitive, whilst analysis of the data is completely automated meaning it can be used by an expert or a complete novice with equal, powerful results.

Its powerful and easy to use genesig q32 software provides a quick start for all genesig kit applications. The software allows straightforward editing of sample and target information, and its automated data analysis calling provides easy interpretation of results.

Key features

As well as being remarkably beautiful, the genesig q16 is technologically powerful. It has no moving parts, making it very robust.

It operates in silence and weighs less than 2kg. It can operate with a PC or Mac but can also operate in stand alone mode with the test data being downloaded on to a USB drive for analysis later.

• Guaranteed results using all genesig kits • Fast DNA and RNA analysis in less than 60 minutes • Quick and easy set-up and run • Automated data analysis result calling • Small and robust instrument • Exceptional value for money

A complete solution

genesig Real-Time PCR Kit compatible

The genesig q16 can be used with regular lab equipment and DNA extraction systems. But if you don’t have a lab, the genesig Easy Labin-a-Box and the genesig Easy DNA/RNA Extraction Kit will provide all that you need to complete your molecular testing set-up.

All genesig Real-Time PCR Detection Kits are suitable for use on the genesig q32 instrument, including Standard, Advanced, and Easy Kit formats, providing the ultimate in flexibility.

Ground-breaking technology

24

REAL-TIME PCR INSTRUMENTS CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-genesig-q16

genesig q16 Real-Time PCR Instrument

1

Z-genesig-q32

genesig q32 Real-Time PCR Insturment

1

25

genesig® Kits

Optimised qPCR detection kits More than 550 genesig® kits

Covering a range of pathogen groups

The genesig kit range includes over 550 kits for human, vet, and food pathogens, as well as food speciation testing. All genesig kits include a positive control and resuspension buffers, and they all have an identical running protocol. The kits come freeze-dried so that they can be shipped at room temperature for fast delivery to anywhere in the World.

Human pathogens kits: HBV, HCV, HIV, CMV, RSV, Chlamydia trachomatis, Neisseria gonorrhoeae, Dengue and many more.

• Rapid detection within 90 minutes • Sensitive to <100 copies of target • Accurate controls to confirm extraction, run and reaction validity • Easy to use for all user experience levels • Fast development of new kits on demand • Supplied lyophilised with no cold chain shipping

genesig Easy Kits The genesig Easy qPCR detection kits are specially optimised for the genesig q16 and are also ideal for use on the genesig q32.

Human pathogen CE-IVD Kits: BKV, EBV, Zika and more coming soon.

Veterinary and aquaculture pathogen kits: ASFV, Bovine TB, Foot and Mouth disease, Taylorella equigenitalis, Avian influenza, Salmon gill poxvirus and many more.

Food pathogen and speciation kits: Listeria, Salmonella, Legionella, Horse speciation, Pork speciation and many more.

Multiplex kits: For a variety of human infections including transplant, STD, respiratory, hepatitis, and blood-borne; as well as for the veterinary infection equine strangles.

OnDemand kits: If your target of interest is missing from our huge menu we can develop a new test for you on demand in just 6 weeks.

Open qPCR Platform Kits We also offer the genesig Standard and Advanced qPCR detection kits which are open qPCR platform format, for use on any qPCR instrument. To see our full range of kits, please visit our website and if you still cannot find a specific pathogen of interest then we can make new kits OnDemand.

Kits contain copy number positive controls to allow for precise copy number determination, an internal extraction control to give detailed insight in to the success of your extraction process.

26

More than 550 genesig kits available!

27

Precision®PLUS qPCR Master Mix

Primerdesign real-time PCR reagents are manufactured to the highest standards. Free samples are available for validation purposes, please enquire.

Amplification graph demonstrating reliable and uncompromised detection of 4 targets using oasigTMPLEX Lyophilised qPCR Master Mix Amplification 14 12 10

RFU (10^3)

High quality, robust 2X qPCR master mix at an exceptionally low cost. The core components are a hot start Taq polymerase enzyme with a magnesium chloride based buffer. Stabilisers and preservatives ensure that multiple freeze-thaw cycles do not affect the performance. It contains a high fidelity enzyme mix which will provide for accurate quantitative measurements for qPCR.

8 6 4 2

Product features • Huge cost saving • Precise reproducible results • High efficiency enzyme - better data • Robust • Available for all machines (see website for complete list) • SYBR® Green dye added free of charge • Inert blue loading dye added free of charge

0 0

10

20

30

40

Green (VIC) = Target pathogen 1, Orange (ROX) = Target pathogen 2 Blue (FAM) = Target Pathogen 3, Purple (Cy5) = Endogenous Control 4-plex qPCR testing conducted on the Bio-Rad CFX instrument using Primerdesign oasigPLEX Lyophilised qPCR Master Mix. A 4-way multiplex genesig Human Pathogen assay (including endogenous control primer and probe) was used to test template mixtures prepared to model a high titre co-infection sample.

Quadruplicates tests were performed.

The assay demonstrated reliable, uncompromised detection of all 4 targets when used in conjunction with oasigPLEX Lyophilised qPCR Master Mix.

All our PrecisionPLUS master mixes are available with or without SYBR® Green premixed at no extra charge. Simply state your preference when ordering or add “-SY” to the catalogue number for clarity.

28

50

Cycles

PRECISIONPLUS QPCR MASTER MIX CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-PPLUS-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix

1ml; 2ml; 5ml; 10ml; 20ml

Z-PPLUS-R-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix with ROX*

1ml; 2ml; 5ml; 10ml; 20ml

Z-PPLUS-LR-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix with low ROX**

1ml; 2ml; 5ml; 10ml; 20ml

Z-PPLUS-iC-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix for the Bio-Rad

1ml; 2ml; 5ml; 10ml; 20ml

Z-PPLUS-CL-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix for the Roche LightCycler 1.0-2.0

1ml; 2ml; 5ml; 10ml; 20ml

Z-PPLUS-MX-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix for Stratagene MX machines

1ml; 2ml; 5ml; 10ml; 20ml

*Recommended for ABI 7000, 7300, 7700, 7900, StepOne and StepOnePlus - If your machine is not specified please enquire **Recommended for ABI 7500, ViiA7 and QuantStudio - if your machine is not specified please enquire.

Huge cost savings!

29

Precision®PLUS OneStep RT-qPCR Master Mix

This increases the rate of processing and the sensitivity of detection.

The result is an RT step that is completed in less than 10 minutes and is sensitive in a linear range across the whole biological scope from very high (>1 x 109 copies) to very low (<10 copies) RNA copy numbers. An RNase inhibitor is included to protect the sample from possible degradation.

Product features • Convenient, one step, closed tube reaction • Rapid protocol - includes 10 minute RT step • Ideal for RNA pathogen detection • Reduced handling/set up time • Reduced risk of contamination • SYBR® Green dye added free of charge • Inert blue loading dye added free of charge

30

Comparison of Precision®PLUS OneStep RT-qPCR Master Mix vs. other commercially available one step RT-qPCR master mixes Amplification 7000

PrecisionPLUS OneStep = 28.34 Competitor B = 28.57 Competitor T = 28.84 Competitor L = 29.05 Competitor K = 29.16 Competitor Q = 38.40 Competitor R = 39.99

6000 5000

RFU (10^3)

Our PrecisionPLUS OneStep RT-qPCR Master Mix contains all of the required components for a perfect one step real-time PCR analysis in a single reaction mix. The operator simply adds RNA and a primer/ probe mix for a complete, closed tube, one step RNA to Cq reaction.

NanoScript2 is a modified thermostable MMLV enzyme with an optimum working temperature of 55°C and a higher affinity for primer duplex initiation sites.

4000 3000 2000 1000 0 0

10

Amplification curves: FAM channel

20

30

40

Cycles

Amplification of the rat Mmp9 gene from rat RNA. Testing was conducted on the Bio-Rad CFX instrument using the Primerdesign Mmp9 rat gene assay. A generic one-step protocol, with an extended enzyme activation step, was applied to enable parallel testing. The reveres transcription stage was applied as a gradient to ensure all master mixes were utilised appropriately.

PrecisionPLUS OneStep RT-qPCR Master Mix (in dark blue) is compared to 6 competitor master mixes in light blue (listed in order of trace appearance).

PrecisionPLUS OneStep RT-qPCR Master Mix produced highly efficient, consistent and reliable detection in comparison to all of the competitor master mixes.

PRECISIONPLUS ONESTEP RT-QPCR MASTER MIX CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-OSPLUS-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix

1ml; 2ml; 5ml; 10ml; 20ml

Z-OSPLUS-R-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix with ROX*

1ml; 2ml; 5ml; 10ml; 20ml

Z-OSPLUS-LR-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix with low ROX**

1ml; 2ml; 5ml; 10ml; 20ml

Z-OSPLUS-iC-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix for the Bio-Rad iCycler

1ml; 2ml; 5ml; 10ml; 20ml

Z-OSPLUS-CL-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix for the Roche LightCycler 1.0-2.0

1ml; 2ml; 5ml; 10ml; 20ml

Z-OSPLUS-MX-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix for Strategene MX machines

1ml; 2ml; 5ml; 10ml; 20ml

*Recommended for ABI 7000, 7300, 7700, 7900, StepOne and StepOnePlus - If your machine is not specified please enquire **Recommended for ABI 7500, ViiA7 and QuantStudio - if your machine is not specified please enquire.

31

Precision®FAST qPCR Master Mix

Product features • Ultra-fast enzyme • qPCR data in 40 minutes • Available for all machines (see website for complete list) • Minimises primer dimer formation • SYBR® Green dye added free of charge • Inert blue loading dye added free of charge All our PrecisionFAST master mixes are available with or without SYBR® Green premixed at no extra charge. Simply state your preference when ordering or add “-SY” to the catalogue number for clarity.

32

Comparison of Precision®FAST qPCR Master Mix vs. other commercially available fast cycling qPCR master mixes Amplification

PrecisionFAST = 29.79 Cq Competitor K = 30.06 Cq Competitor T = 30.53 Cq Competitor Q = 32.86 Cq Competitor R = 47.93 Cq

25 20

RFU (10^3)

PrecisionFAST qPCR master mix is an ultra-fast, cost-saving mix for qPCR. The 2X mix is designed for rapid cycling protocols that can dramatically shorten run times. The Taq polymerase has been mutated at the active site and has higher affinity for DNA and faster processing. The buffer has been designed for optimum sensitivity and also to reduce primer dimers which are a common artefact of fast processing enzymes.

15 10 5 0 0

10

Amplification curves: FAM channel

20

30

40

50

Cycles

Amplification of the rat Mmp9 gene from rat cDNA. Testing was conducted on the Bio-Rad CFX instrument using the Primerdesign Mmp9 rat gene assay. A generic fast cycling protocol, with an extended enzyme activation step, was applied to enable parallel testing.

PrecisionFAST qPCR Master Mix (in dark blue) is compared to 4 competitor fast master mixes in light blue (listed in order of trace appearance).

PrecisionFAST qPCR Master Mix produced rapid detection whilst maintaining the highest standards of efficiency, consistency and reliability in comparison to the competitor fast master mixes.

PRECISIONFAST QPCR MASTER MIX CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-PFAST-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix

1ml; 2ml; 5ml; 10ml; 20ml

Z-PFAST-R-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix with ROX*

1ml; 2ml; 5ml; 10ml; 20ml

Z-PFAST-LR-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix with low ROX**

1ml; 2ml; 5ml; 10ml; 20ml

Z-PFAST-iC-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix for Bio-Rad machines

1ml; 2ml; 5ml; 10ml; 20ml

Z-PFAST-CL-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix for the Roche LightCycler 1.0-2.0

1ml; 2ml; 5ml; 10ml; 20ml

Z-PFAST-MX-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix for Stratgene MX machines

1ml; 2ml; 5ml; 10ml; 20ml

*Recommended for ABI 7000, 7300, 7700, 7900, StepOne and StepOnePlus - If your machine is not specified please enquire **Recommended for ABI 7500, ViiA7 and QuantStudio - if your machine is not specified please enquire.

qPCR data in ~40 minutes 33

oasig®Lyophilised 2X qPCR and oasig®Lyophilised 2X OneStep RT-qPCR Master Mix High quality, robust 2X qPCR master mix and OneStep RT-qPCR master mix supplied lyophilised.

Amplification plot demonstrating the consistency of oasig™ Lyophilised qPCR Master Mix after repeated freeze thaws

The core components are a hot start Taq polymerase enzyme and a modified MMLV reverse transcriptase enzyme with a magnesium chloride based buffer. Stabilisers and preservatives ensure that lyophilisation does not affect the performance.

• Supplied lyophilised – no cold shipping required • Precise reproducible results • One product works perfectly with all real-time PCR machines

25 20

RFU (10^3)

Product features

Amplification 30

15 10 5 0 0

10

20

30

40

50

Cycles

oasig lyophilised reagents represent a milestone in qPCR technology Their formulation stabilises all of the active components and allows them to be shipped and stored at room temperature. They are stable for more than 18 months at ambient temperatures.

This hugely simplifies the logistics of purchasing, shipping and using the technology. Whether you are in a sophisticated laboratory in Texas or a mobile field hospital in Timbuktu we can supply complete qPCR kit and reagent packages to your door quickly and cheaply via standard shipping methods without the need for dry ice or a cold chain of any sort.

The performance of the reagents is second to none. We are confident that you will find excellent data quality and even see an improvement in data quality versus many traditional frozen master mixes.

34

Blue: oasig Lyophilised qPCR Master Mix Light Blue: oasig Lyophilised qPCR Master Mix after 10 freeze-thaw cycles

Testing was conducted on the BioRad CFX instrument using a selection of Primerdesign rat gene assays and standard genesig cycling conditions.

Two separate tubes were used to amplify the rat genes from rat cDNA. The first tube was subjected 10 freeze-thaw cycles (freezing conducted at -20°C) whilst the second served as a positive control (kept in -20°C storage).

No significant effect on the performance of oasig Lyophilised qPCR Master Mix was observed after 10 freeze-thaws. This demonstrates suitability of oasig Lyophilised qPCR Master Mix for multiple freeze-thaw uses and illustrates that optimal performance levels will be maintained under these conditions.

OASIG LYOPHILISED MASTER MIX CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-oasig-standard-150

oasig Lyophilised 2X qPCR Master Mix

150 reactions

Z-oasig-onestep-150

oasig Lyophilised 2X OneStep RT-qPCR Master Mix

150 reactions

Supplied lyophilised â&#x20AC;&#x201C; no cold shipping required

35

oasig®PLEX Lyophilised qPCR Master Mix

The oasigPLEX Master Mix contains optimised enzyme levels and our proprietary enzyme-buffer system to maximise performance of multiplex assays. Included in our oasigPLEX qPCR Master Mix is ampliSOLVE®, an innovative solution that will enhance qPCR efficiency through artefact removal. The function of ampliSOLVE is to remove amplicon contamination, resulting in an enhanced efficiency and performance of the multiplex qPCR reaction.

The qPCR master mix is designed for rapid cycling protocols and contains an antibody mediated Hot-Start mechanism which releases more active enzyme and requires a much shorter activation time.

The freeze-dried product is stable at ambient temperatures for at least 18 months and can be conveniently shipped and stored at room temperature.

Product features • Optimised for multiplex applications • Higher throughput • Reduced shipping costs • ampliSOLVE for artefact removal • Supplied lyophilised - no cold chain shipping required • Experiment set up at room temperature

36

Amplification graph demonstrating reliable and uncompromised detection of 4 targets using oasigTMPLEX Lyophilised qPCR Master Mix Amplification 14 12 10

RFU (10^3)

Primerdesign oasigPLEX Lyophilised qPCR Master Mix is a freezedried speciality 2X master mix optimised for use in multiplex qPCR. With set up possible at room temperture, it means working from an ice box is not required making experiment set up that much easier.

8 6 4 2 0 0

10

20

30

40

50

Cycles

Green (VIC) = Target pathogen 1, Orange (ROX) = Target pathogen 2 Blue (FAM) = Target Pathogen 3, Purple (Cy5) = Endogenous Control 4-plex qPCR testing conducted on the Bio-Rad CFX instrument using Primerdesign oasigPLEX Lyophilised qPCR Master Mix. A 4-way multiplex genesig Human Pathogen assay (including endogenous control primer and probe) was used to test template mixtures prepared to model a high titre co-infection sample.

Quadruplicates tests were performed.

The assay demonstrated reliable, uncompromised detection of all 4 targets when used in conjunction with oasigPLEX Lyophilised qPCR Master Mix.

PRECISIONULTRA ONESTEP RT-QPCR MASTER MIX CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-oasigPLEX-150

oasigPLEX Lyophilised qPCR Master Mix

150 reactions

Optimised for multiplex applications

37

oasig®PLEX Lyophilised OneStep RTqPCR Master Mix Primerdesign oasigPLEX Lyophilised OneStep RT-qPCR Master Mix is a freeze-dried speciality 2X master mix optimised for use in multiplex RT-qPCR. The qPCR master mix is designed for rapid cycling protocols and contains an antibody mediated Hot-Start mechanism which releases more active enzyme and requires a much shorter activation time.

Amplification graph demonstrating reliable and uncompromised detection of 4 targets using oasigTMPLEX Lyophilised OneStep RT-qPCR Master Mix Amplification 14 12

The freeze-dried product is stable at ambient temperatures for at least 18 months and can be conveniently shipped and stored at room temperature.

Product features • Optimised for multiplex applications • Higher throughput • Reduced shipping costs • ampliSOLVE for artefact removal • Supplied lyophilised - no cold chain shipping required • Suitable for both RNA and DNA • Experiment set up at room temperature

38

RFU (10^3)

10

The oasigPLEX Lyophilised OneStep Master Mix contains optimised enzyme levels and our proprietary enzyme-buffer system to maximise performance of multiplex assays. Included in our oasigPLEX Lyophilised OneStep RT-qPCR Master Mix is ampliSOLVE®, an innovative solution that will enhance qPCR efficiency through artefact removal. The function of ampliSOLVE is to remove amplicon contamination and reduce primer dimer formation.

Resulting in an enhanced efficiency and performance of the RTqPCR reaction.

8 6 4 2 0 0

10

20

30

40

50

Cycles

Blue (FAM) = Target Pathogen 1, Green (VIC) = Target pathogen 2, Orange (ROX) = Target pathogen 3, Purple (Cy5) = Endogenous Control 4-plex qPCR testing conducted on the Bio-Rad CFX instrument using Primerdesign oasigPLEX Lyophilised OneStep RT-qPCR Master Mix. A 4-way multiplex genesig Human Pathogen assay (including endogenous control primer and probe) was used to test template mixtures prepared to model a high titre co-infection sample.

Quadruplicates tests were performed.

The assay demonstrated reliable, uncompromised detection of all 4 targets when used in conjunction with oasigPLEX Lyophilised OneStep RT-qPCR Master Mix.

PRECISION HRM MASTER MIX CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-oasigPLEX-OS-150

oasigPLEX Lyophilised OneStep RT-qPCR Master Mix

150 reactions

Suitable for both RNA and DNA

39

BrightWhite Real-Time PCR qPCR Plates Product features

The best possible real-time PCR is performed using white, opaque qPCR plates. As fluorescence is the crucial output from a realtime PCR reaction, it is essential that as much of the fluorescent information available as possible is captured. Clear plastics spray light in all directions. BrightWhite qPCR plates channels all of the fluorescent output from your reaction straight back to the detector.

• Precisely moulded for specific machines • White opaque plastic gives brighter, better data • Extreme uniformity of wall thickness • Truly flat plates reduce well-to-well variability • Cost effective pricing

When using white plastic we recommend using a PCR inert blue dye, which makes visualising your pipetting much easier on white plastic.

Overall BrightWhite vs Clear Plastic Amplification Plot 7,000,000

Brightwhite Clear Plastic

6,000,000

Rn

5,000,000 4,000,000 3,000,000 2,000,000 1,000,000 0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

Cycle

40

28

30

32

34

36

38

40

41

42

44

46

48

50

BRIGHTWHITE REAL-TIME PCR PLATES CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-BW-96AB1

96-well plates for ABI machines: ABI7000, ABI7300, ABI7500, ABI7700, ABI7900

10 plates

Z-BW-FAST

96-well plates for ABI7500 Fast, ABI7900 Fast and ABI StepOne plus machines

10 plates

Z-BW-96GEN

96-well plates for Bio-Rad, Stratagene, Eppendorf and other machines

10 plates

Z-BW-96480

96-well plates for the Roche LightCycler 480 and Bio-Rad CFX machines

10 plates

Z-BW-384GEN

384-well plates for a wide variety of machines, e.g. Applied Biosystems, Bio-Rad, Eppendorf, Roche, MJ Research and Whatman Biometra

10 plates

Z-BW-ECO

48-well plates for the Illumina Eco and PrimePro 48

40 plates

Z-BW-ADVSEAL

Optical adhesive seals for BrightWhite 96 and 384 well plates

12 seals

Z-BW-8STRIP

Optical 8 strip cap seals for BrightWhite 96 well plates

120 strips

Z-BW-ECOSEAL

Optical adhesive seals for BrightWhite 48 well plates

40 seals

At least 5 x more fluorescent output vs. clear plastic

41

genesig® Easy Tips genesig Easy tips for pipettes have been robustly assessed by Primerdesign and found to represent optimal level of performance in terms of pipetting accuracy and precision.

Product features • Sterile • Certified free of RNase/DNase, Human DNA, PCR inhibitors and Pyrogens • Micropore filter technology • CE-IVD mark for laboratory use

GENESIG® EASY PIPETTE TIPS CATALOGUE NO.

PRODUCT DESCRIPTION

BOX SIZE

Z-genesigEASY-P20

Pipette tips for up to 20ul, box of 10x96

Box of 10 x 96

Z-genesigEASY-P200

Pipette tips for up to 200ul, box of 10x96

Box of 10 x 96

Z-genesigEASY-P500

Pipette tips for up to 500ul, box of 10x96

Box of 10 x 96

Z-genesigEASY-Pipettes

Set of 4 genesigEASY pipettes, 5ul, 10ul, 200ul and 500ul

Set of 4 - 5ul, 10ul, 200ul and 500ul

42

RNase/DNase Free Water suitable for molecular biology

RNase or DNase contamination in the water used for molecular biology assays can lead to experimental errors or inconsistencies.

Our high quality RNase/DNase Free Water provides peace of mind when conducting sensitive molecular biology assays such as qPCR.

Product features • Ultrapure molecular biology grade water • RNase and DNase free • DEPC-treated, autoclaved, and filtered through a 0.2-micron filter • Suitable for applications such as qPCR

Ultrapure molecular biology grade water RNASE/DNASE FREE WATER CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-RNase/DNase free water

RNase/DNase Free Water

15ml (10 x 1.5ml aliquots)

43

BioBank Control cDNA The ideal positive control for your experiment. The BioBank is a high quality source of cDNA validated for use in real-time PCR experiments. The cDNA is reverse transcribed from high quality, DNase treated RNA, from a variety of tissues and cell cultures, using an optimised blend of oligo(dT) and random nonamer primers.

BioBank cDNA is therefore free of genomic DNA and PCR inhibitors and covers the widest possible range of RNA and mRNA transcripts in the specified tissue or cell line. BioBank cDNA is useful for expression profiling of newly identified genes and also as a positive control for real-time PCR. Positive control primer/probe are supplied with the kits which detect 18S ribosomal RNA. The 18S control primers are especially formulated to work under all recommended real-time PCR cycling conditions; i.e. TaqMan® cycling and SYBR® Green cycling conditions will all work perfectly with the kit.

Product features • High quality cDNA • Validated for use in real-time PCR • Free from PCR inhibitors • Guaranteed signal • Reverse transcribed using optimal oligo(dT)/random nonamer blend

44

Kit contents • 250ng of gDNA-free cDNA (minimum 25 reactions) • Positive control 18S primer and probe set • RNase/DNase free water

BIOBANK CONTROL cDNA HUMAN CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-cDNA-hu-ge Z-cDNA-hu-ti Z-cDNA-hu-cl Z-cDNA-hu-pr

Generic BioBank cDNA pooled from a collection of human tissues and cells BioBank cDNA from a specific human tissue* BioBank cDNA from a specific human cell line* BioBank cDNA from a specific primary cell*

250ng 250ng 250ng 250ng

CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-cDNA-mo-ge Z-cDNA-mo-ti

Generic BioBank cDNA pooled from a collection of mouse tissues and cells BioBank cDNA from a specific mouse tissue*

250ng 250ng

MOUSE

Estimated despatch: 4-6 weeks * Please contact us or visit our website for the latest list of available tissues/cell lines and primary cells.

45

Real-Time PCR Internal Control Kit Negative test results are only valid when the suitability of the reagents, template and protocols have also been demonstrated.

The Primerdesign Real-Time PCR Internal Control Kit contains either control RNA or DNA. This can be spiked in to your sample prior to nucleic acid extraction or diluted and spiked directly into your realtime PCR reaction. A positive signal of around Cq 25 indicates that your experimental procedure is working well.

The Internal Control kit can be multiplexed or used in parallel with your test samples. The assay is primer limited and based on an obscure nucleic acid sequence so will not therefore interfere with the detection of your target sequence.

Product features • Perfect internal control • Hydrolysis (TaqMan® style) probe • Available with a wide range of fluorophores • Primer limited to prevent interference with target gene primers

Kit contents • Lyophilised internal control primers/hydrolysis (TaqMan style) probe (150 reactions) • Internal control DNA or RNA template • RNase/DNase free water

46

REAL-TIME PCR INTERNAL CONTROL KIT CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-INT-RNA-FAM Z-INT-RNA-CY5 Z-INT-RNA-YY

RNA Internal PCR control with FAM dye RNA Internal PCR control with Cy5 dye RNA Internal PCR control with Yakima Yellow (VIC substitute) dye

150 reactions 150 reactions 150 reactions

CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-INT-DNA-FAM Z-INT-DNA-CY5 Z-INT-DNA-YY Z-INT-DNA-CY5-LL

DNA Internal PCR control with FAM dye DNA Internal PCR control with Cy5 dye DNA Internal PCR control with Yakima Yellow (VIC substitute) dye Suitable for use with ultra-fast master mixes

150 reactions 150 reactions 150 reactions 150 reactions

DNA

Perfect internal control 47

Positive Control Template for Standard Curve This kit provides for a standard curve with a dynamic range from 106 to 10 copies. Under ideal PCR conditions, 10 copies of the target will be detected. Primerdesign guarantees that a minimum of 100 copies will be detected.

Product features • Provides a positive standard curve for qPCR experiments • Wide dynamic range from 1 million copies to 10 copies • Available for: human, mouse, rat, and other species

Kit contents • Positive control template for standard curve • emplate preparation buffer • RNase/DNase free water

48

Perfect internal control

POSITIVE CONTROL TEMPLATE FOR STANDARD CURVE CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-HU-STD Z-MO-STD Z-RA-STD Z-ANY-STD

Human positive control template Mouse positive control template Rat positive control template Other species positive control template

150 reactions 150 reactions 150 reactions 150 reactions

49

Genomic DNA Detection Kits Quantification of genomic DNA is useful for forensic research and also as a normalising signal for quantitative PCR and cell counting.

The Primerdesign genomic DNA detection assays are sensitive down to 25pg of genomic DNA.

Product features

No more RT negative controls: The Primerdesign genomic DNA (gDNA) detection assays targets only gDNA and not cDNA and is the perfect alternative to the use of a ‘reverse transcription negative control’.

Detection kit contents

These primer sets are designed against single copy sequences within the genome. This sequence is not within any known gene.

Quantification kit contents

• Genomic DNA specific • Sensitive to pg quantities of DNA • Novel replacement for ‘RT negative control’ • Optimised primer/hydrolysis (TaqMan®-style) probe mix (150 reactions) RNase/DNase free water • Optimised primer/hydrolysis (TaqMan-style) probe mix (150 reactions) • gDNA positive control template • Internal extraction control DNA • Internal extraction control primer/probe mix (150 reactions) • RNase/DNase free water

GENOMIC DNA DETECTION KITS DETECTION KIT CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-gDNA-hu Z-gDNA-mo Z-gDNA-ra

Human genomic DNA detection kit Mouse genomic DNA detection kit Rat genomic DNA detection kit

150 reactions 150 reactions 150 reactions

CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-gDNA–hu-q Z-gDNA-mo-q Z-gDNA-ra-q

Human genomic DNA quantification kit Mouse genomic DNA quantification kit Rat genomic DNA quantification kit

150 reactions 150 reactions 150 reactions

QUANTIFICATION KIT

50

Y Chromosome Detection Kits Quantification of Y chromosome DNA is useful for forensic research.

The Primerdesign Y chromosome DNA detection assays are sensitive down to 25pg of target DNA.

Product features • Y chromosome specific • Sensitive to pg quantities of DNA

Detection kit contents

Sensitive to pg quantities of DNA

• Optimised primer/probe mix (150 reactions) • RNase/DNase free water

Quantification kit contents • Optimised primer/probe mix (150 reactions) • gDNA positive control template • Internal extraction control DNA • Internal extraction control primer/probe mix (150 reactions) • RNase/DNase free water

Y CHROMOSOME DETECTION KITS WITH TAQMAN® STYLE PROBE CATALOGUE NO.

PRODUCT DESCRIPTION

KIT SIZE

Z-yDNA-hu-DD Z-yDNA-hu-DD-q

Human Y chromosome DNA detection kit with hydrolysis (TaqMan style) probe Human Y chromosome DNA quantification kit with hydrolysis (TaqMan style) probe

150 reactions 150 reactions

51

Molecular Diagnostics (MDx) Assay Development Services Primerdesign are experts in molecular diagnostic assay development.

We have conducted many successful projects with leading diagnostic and biotechnology companies. Our B2B division partners with diagnostic companies to provide the following services: • Assay design • Prototype testing • Assay pre-feasibility and optimisation • Assay validation (analytical and clinical) • Stability studies • Technical file construction and regulatory approval • Kit manufacturing (ISO13485) We routinely bring customer’s platforms in-house to optimise assays to ensure maximum performance. We can also optimise assays in an open format to work on all MDx platforms.

We can manufacture molecular diagnostic assays in freeze-dried, air-dried and liquid formats.

Primerdesign products

Primerdesign’s impressive >550 genesig® diagnostic product range and proprietary reagents (master mixes and positive controls) are available on either an OEM basis or for distribution.

For more information please visit www.primerdesign.co.uk.

STEP ONE

STEP TWO

STEP THREE

STEP FOUR

DESIGN & FEASIBILITY STUDIES

DEVELOPMENT & VALIDATION

ASSAY TRANSFER & PRODUCTION

QUALITY & PRODUCT RELEASE

• Experts in the design of complex molecular diagnostic kits • Assay optimisation, verification and validation • Lyophilisation of final product

52

Primerdesign has ISO13485 certified manufacturing facilities and has substantial experience in manufacturing finished products that are ready for distribution.

• Extensive kit optimisation and validation • Creation of design history files

• Transfer of assays from development to production • Batch validation • ISO13485 compliant manufacturing • Finished product ready for distribution

• Experienced QA department • Stringent product QC analysis • Fully documented compliant processes • ISO13485 certified • Regulatory approval of MDx Assays

Proven track record in creating ‘market ready’ assays

53

Technical Bulletins We believe that we have failed you if your experiments fail. We love it when people get fantastic data and make exciting discoveries using our kits and reagents. So we place a lot of emphasis on making sure that every experiment you do with our kits gets you meaningful, scientifically accurate data.

So this section of our catalogue is designed to help you succeed in your real-time PCR and to avoid some of the common pitfalls. We have tried to walk you through some of the classical queries and questions that our customers run in to.

By sharing this knowledge and experience with you we hope that your understanding of real-time PCR will improve and so will your data.

More training and education We frequently spend time sharing our expertise in real-time PCR amongst the scientific community. If you would be interested in one of our team coming to your organisation to present a genuinely helpful, free, lunch-time seminar on realtime PCR then please do contact us: enquiry@primerdesign.co.uk Alternatively check out our free online webinars at www.primerdesign.co.uk/learn/free-qpcr-webinars

54

Technical bulletins 56

01 Real-Time PCR Basic Principles

60

02 Probe vs SYBR® Green

62 03 RNA Extraction and Sample Preparation 64

04 Reverse Transcription

66

05 Designing High Quality Primers

68 06 Troubleshooting when using Intercalculating Dyes 72

07 Normalisation

74 08 Standard Curves – Measuring Primer Efficiency 78 09 The Delta Cq method – Relative Quantification 80 10 Standard Curves – Measuring Absolute Copy Number 82

11 The MIQE Guidelines

55

Technical bulletin 01

Real-Time PCR Basic Principles PCR or the Polymerase Chain Reaction has become the cornerstone of modern molecular biology the world over. Real-time PCR is an advanced form of the Polymerase Chain Reaction that maximizes the potential of the technique.

To begin the reaction the temperature is raised to 95°C. At this temperature all double stranded DNA is “melted” in to single strands:

To understand real-time PCR it is easier to begin with the principles of a basic PCR: PCR is a technique for amplifying DNA. There are 2 reasons why you may want to amplify DNA. Firstly you may want to simply create multiple copies of a rare piece of DNA. For example a forensic scientist may want to amplify a tiny piece of DNA from a crime scene. More commonly however you may wish to compare 2 different samples of DNA to see which is the more abundant. DNA analysis requires amplification in order for there to be enough DNA to give a detectable signal for quantification. If you amplify both samples at the same rate, you can calculate which sample had the highest copy number of the target of interest to begin with.

A thermostable polymerase enzyme drives a PCR. This polymerase will synthesize a complementary sequence of bases to any single strand of DNA providing it has a double stranded starting point.

This is very useful because you can choose which gene you wish the polymerase to amplify in a mixed DNA sample by adding small pieces of DNA complimentary to your gene of interest. These small pieces of DNA are known as primers because they prime the DNA sample ready for the polymerase to bind and begin copying the gene of interest.

During a PCR, changes in temperature are used to control the activity of the polymerase and the binding of primers.

56

The temperature is then lowered to ~60°C. This allows the primers to bind to your gene of interest:

Thus the polymerase has somewhere to bind and can begin to copy the DNA strand:

The optimal temperature for the polymerase to operate is 72°C so at this point the temperature is sometimes raised to 72°C to allow the enzyme to work faster.

There are now twice as many copies of your gene of interest as when you started: This temperature change is repeated through around 40 ‘cycles’.

Thus one copy becomes 2, 2 become 4, 4 become 8, and so on until billions of copies are created.

After amplifying your gene it is possible to run the amplified DNA out on an agarose gel and stain it with a dye which makes it visible.

The brighter the visible band, the more copies of your target you have created.

Real-Time PCR This same principle of amplification is employed in real-time PCR. But instead of looking at bands on a gel at the end of the reaction, the process is monitored in “real-time”. Literally, the reaction is placed in to a real-time PCR machine that watches the reaction occur with a camera or detector.

There are a many different techniques that are used to allow the progress of a PCR reaction to be monitored but they all have one thing in common. They all link the amplification of DNA to the generation of fluorescence which can simply be detected with a camera during each PCR cycle. These different techniques are discussed on page 60. Hence, as the number of gene copies increases during the reaction, so the fluorescence increases.

57

Technical bulletin 01

Real-time PCR has many benefits over the old fashioned approach: Firstly it gives you a look in to the reaction. You can literally see which reactions have worked well and which have failed.

• The efficiency of the reaction can be precisely calculated.

• There is also no need to run the PCR product out on a gel after the reaction as the melt curve analysis effectively does this for you.

• The greatest advantage of all however, is that real-time PCR data can be used to perform truly quantitative analysis of gene expression. In comparison, old fashioned PCR was only ever semiquantitative at best.

•

58

59

Technical bulletin 02

Probe vs SYBR® Green

Choosing the correct detection chemistry for your experiment Introduction When designing a real-time PCR experiment a significant decision is choosing the correct detection chemistry for your application. There are two major options to choose from. Most people use either an intercalating dye (e.g. SYBR® Green) or a hydrolysis probe based detection solution (e.g. TaqMan®). Both technologies are designed to generate fluorescence during the PCR, which allows your real-time PCR machine to monitor the reaction in “real time”.

SYBR® Green (or other intercalating dye) SYBR® Green is by far the most commonly used intercalating dye.

There are others available but it’s very likely that SYBR® Green is the one you have heard of. All of these dyes operate via a simple mechanism.

60

The dye is fluorescent in it’s own right but in the presence of double stranded DNA, the dye intercalates with (binds in to) the DNA double helix. This alters the structure of the dye and causes it to fluoresce more. So very simply as the PCR creates more DNA, more dye can bind and more fluorescence is generated.

Hydrolysis (TaqMan® style) Probes Probes are fluorescently labelled DNA oligonucleotides. They are designed to bind downstream of one of the primers during the PCR reaction and to give a fluorescent signal during the reaction. The 5’ end of the probe is labelled with a fluorescent reporter molecule.

FAM is a green reporter and is the most commonly used but there are others such as VIC, JOE, CY5 etc which emit light at different wave lengths and can be read through separate detecting channels.

On the 3’ end of the probe is a quencher molecule. This is another molecule that effectively quenches the output from the reporter.

Therefore, when the reporter and quencher are physically close to one another the overall level of fluorescent output is low.

During the PCR the probe binds downstream of the primer. The probe is then cleaved by the polymerase enzyme during the reaction.

By cleaving the probe the reporter and quencher are separated which means that the quencher no longer has its effect over the reporter and the level of fluorescence increases. This means that with every cycle of PCR more probe is cleaved and more fluorescence is generated.

Things to Consider: Cost Cost is an important part of any experimental design. It is more expensive to use a hydrolysis probe than an intercalating dye.

This means that if you have an experiment looking at lots of different genes or targets then you will probably want to choose an intercalating dye as your detection chemistry. However, if you only have a few targets of interest and you know that they will be your focus, you should choose to use a hydrolysis probe.

Things to Consider: Specificity Hydrolysis probes: Overall, hydrolysis probes give more convenient data to work with than intercalating dyes. This is because of their inherent specificity. When you get a signal from a hydrolysis probe you can be sure that the signal has come from genuine amplification of your target sequence. It is only possible to get a signal when the primers and the probe bind in the correct place, to the correct target.

So no post run analysis is required to confirm the correct target has been amplified. This means the data is inherently reliable and simple to work with.

Intercalating dyes: the weakness of intercalating dyes is that they are non specific. If your PCR amplifies the wrong target, or even

more than one target, you will still get an amplification plot that looks identical to a genuine signal. Intercalating dyes will bind to and report on any double stranded DNA that is formed during the reaction regardless of what it is. This means that additional analysis is required to be certain of your results. This analysis is in the form of a ‘melt curve’ (sometimes called ‘dissociation curve’). Melt curves are discussed in detail on page68. They require more time and more experience to interpret correctly. It is crucial to interpret the melt curve along side the amplification plot in order to work with your data.

Things to Consider: Target Abundance Because of its inherent lack of specificity, intercalating dyes are less effective when your target of interest is rare. When a target is rare the primers are more likely to form primer dimers or to bind to the wrong target. Even the very best primers will eventually form a primer dimer and give a false signal after enough cycles of PCR if no authentic target of interest is present. However, when there is plenty of your target of interest present then intercalating dyes work very nicely.

So as a rule of thumb, if your target of interest is rare (Cq values >30), then using an intercalating dye may well be troublesome and a hydrolysis probe is recommended. But if your target of interest is abundant (Cq values <30) then using an intercalating due will be perfectly sufficient.

Related products Custom Gene of Interest Real-Time PCR Assays PrecisionPLUS Master Mix

pg 22-23 pg 28-29

61

Technical bulletin 03

RNA Extraction and Sample Preparation Introduction Many experiments centre around extracting RNA from a sample (cells, biopsy, blood etc) then performing a reverse transcription reaction to create cDNA to act as a template for the real-time PCR reaction. Successful extraction of pure, high quality RNA is essential to achieve good quality data. Put simply, poor RNA will guarantee you poor quality data.

Columns or Phenol?

The most common RNA extraction protocols use either small anion exchange columns (e.g. QIAGEN columns) to facilitate RNA extraction or use a phenol based precipitation method (e.g. Trizol). Both can work well but the column based kits are far more convenient and tend to result in a higher quality and yield of RNA. The phenol based methods are cost effective but are fiddly and rely on a high degree of benchside experience and skill in order to yield good quality RNA. Our recommendation is therefore is to use a column based solution.

Invest in good RNA at this stage and everything else downstream will become much more successful.

RNA Quality It is impossible to stress enough how important good quality RNA is for successful real-time PCR. RNA is inherently fragile. Your skin is covered with RNases designed to protect you from RNA viruses.

These will destroy RNA in the blink of an eye. So it is essential to follow good laboratory practices when working with RNA. Set up a designated RNA working area with a PCR hood. Wear a lab coat and gloves, and always keep RNA on ice when not frozen.

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Once you have extracted RNA you need to be certain it is of a good quality before you proceed with a relatively expensive real-time PCR protocol. If your RNA is of a poor quality then you should discard it and start again as it will only yield poor quality data downstream.

To check RNA quality there are a few options available. Nanodrop machines (or similar) are commonly found in many laboratories these days and will tell you about the quantity and the purity of your RNA. However, they do not give any information about RNA integrity which is equally critical. For this, the ideal tool is a Bioanalyser (micro fluidic electrophoresis machine). Several such machines are available on the market. They work by assessing the relative abundance and integrity of 18S and 28S ribosomal RNA and will give you a simple quality score for your RNA. If you do not have access to one of these then you can simply run a small amount of your RNA on a 1% agarose gel and stain with ethidium bromide (or equivalent). When viewed under UV, good quality RNA will show two

distinct bands (18S and 28S ribosomal RNA), where as degraded RNA will appear as a smear on the gel leading from the ribosomal bands to the smaller fragments at the top of the gel.

DNase Treatment During RNA extraction it is very likely that you will incidentally co-extract gDNA alongside the RNA. This will cause problems downstream if it is not removed so a quick and simple DNase treatment should be carried out. This needs to be done thoughtfully as it is crucial not to damage the RNA during this process. We recommend the use of our DNase treatment kit (see page 8).

Related products genesigÂŽ Easy DNA/RNA Extraction Kit Precision DNase kits

pg 04-05 pg 08-09

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Technical bulletin 04

Reverse Transcription Introduction Reverse transcription (RT) is a simple and elegant process that converts RNA into cDNA thus creating a suitable template for real-time PCR. The RNA is primed by the addition of single stranded DNA primers that bind to the RNA. Where primed, the reverse transcriptase enzyme can bind to the RNA and synthesise a complimentary strand made of DNA. The best reverse transcription protocols use thermostable enzymes capable of working at relatively high temperatures (approx 55C°). At these higher temperatures enzymes work faster and there is less tangling of the RNA. This results in a faster more efficient reaction.

Priming Strategy It is important to put some thought into the priming strategy that you use in your RT reaction. Different strategies are suitable for different applications.

Random primers Random primers are short (typically 6-9 bases in length) DNA primers of a random sequence. They are designed to bind all over your RNA allowing reverse transcription of the entire sequence. A feature of random primers is that they will prime ribosomal RNA as well as messenger RNA. For most people it is not necessary to prime rRNA as it is mRNA that is the template of interest. rRNA accounts for around 90% of all the RNA in an extraction. This means that if you are not interested in rRNA targets, then 90% of the enzyme activity is wasted in

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this instance, resulting in an inefficient reverse transcription of the mRNA you are interested in. In this scenario it is better to use one of the alternative priming strategies described below. Random primers are suitable however, if you are looking to measure rRNA expression, working with partially degraded RNA or working with bacteria and viruses where the mRNA do not have polyA tails.

Oligo(dT) primers Oligo(dT) primers are simply a short string of T’s. These are designed to bind to the polyA tail of mRNA, thus creating a priming site at the 3’ end of the gene to which the RT enzyme can bind and begin reverse transcription. The advantage of this approach is that it is mRNA specific. This makes for a more efficient RT reaction in most cases as the enzyme activity is focused on mRNA and ignores the abundant rRNA. Typically a switch to Oligo(dT) priming can result in a 2-4 fold increase in RT efficiency and this improves your subsequent real-time PCR data.

Oligo(dT) primers are not suitable if your RNA is degraded, if you are hoping to measure rRNA expression, or when working with species where the mRNA does not have a polyA tail.

Gene specific primers Gene specific priming is the gold standard way to perform reverse transcription. An RT primer is designed at the same time as your real-time PCR primers and binds specifically to the same region as your real-time PCR primers. When this primer is used for the RT step then the enzyme activity is focused only on your gene of interest resulting in a maximally efficient reverse transcription reaction. This approach is particularly useful if your gene of interest is rare and you are trying to achieve the maximum possible signal strength to improve your data.

The perceived downside of gene specific RT priming is that you can only measure one target of interest in the resultant cDNA. i.e. you cannot return to the cDNA at a later date and study the expression of other genes. This is true if only one gene specific primer is used.

But there is no reason why up to 20 or 30 gene specific primers for all your targets of interest cannot be pooled and then used as an RT priming strategy. This still focuses the enzyme activity on only a few targets. Compared to other random priming strategies that prime thousands of transcripts, this is still a very focused approach and will result in better real-time PCR data.

Related products Precision RT kits

pg 10-11

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Technical bulletin 05

Designing High Quality Primers (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

High quality primers are critical to obtaining good quality data. The reverse statement is also true. Poor primers will always produce inconsistent and poor quality data.

Priming Specificity

Designing and optimising high quality primers can be a time consuming and onerous task. We are the best in the World at designing primers so the simplest and often most cost effective route to take is to ask us to design and optimise your primers for you. We can have your perfect primers back to you in as little as two weeks.

The priming specificity can only be tested experimentally by performing a real-time PCR reaction on your cDNA. To do this you need to include an intercalating dye ( e.g. SYBR® Green) in the reaction and perform a melt curve analysis (see page 70). A single melting peak of the correct predicted temperature indicates that only the one PCR product of interest has been amplified by the primers.

If you do decide to have a crack at designing primers yourself there are a wide range of issues that need to be given careful consideration in order to meet the high standards for quantitative PCR. This is a brief overview of the key points.

Priming Efficiency

In order to succeed, primers must be: • • •

Specific to one target Active in amplifying only one PCR product Highly efficient at initiating PCR

Bioinformatics In order to do quantitative PCR for a single target, the primers must bind only to that one target. This may sounds very obvious but in practice there are often closely related transcripts or pseudo-gene transcripts present in the genome that must be excluded from detection. The best way to analyse the bioinformatics specificity of the primers is to do sequence comparison with the entire amplicon (the full length of the PCR product which includes the primers). The most useful resources are the publically available NCBI databases

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Priming efficiency can be measured by performing a standard curve (see page 76). However, ensuring good performance is a matter of good primer design. Poor priming efficiency is caused by poor primer to template binding kinetics and by priming artefacts that can occur with poorly designed primers. The biggest factor that can affect the efficient binding of primers to the target template is the thermodynamic structure of the single DNA strand to which the primers bind. Single stranded DNA strands have the intrinsic property to fold into complex hairpin structures. If these structures occur strongly at the site of the primer binding then the efficiency of binding is reduced leading to poor PCR priming efficiency. Ideally the template structure should be modelled using the latest software.

However some fairly crude online tools are available for free. The Mfold program can be used to indicate if template structure is likely to be a problem (http://mfold.rna.albany.edu/?q=mfold/DNAFolding-Form).

Testing your Primers Historically in PCR people have spent a lot of time optimising reaction conditions to make bad primers work better. We donâ&#x20AC;&#x2122;t believe that a bad set of primers should be fixed in this way. It often leads to a more complicated and harder to interpret set of data.

The best advice we can you give you is that if your primers are not working well for you with minimal optimisation then throw them away and start over. Alternatively let us use our expertise and state of the art processes to deliver a high quality validated design straight into your reaction.

Related products Custom Gene of Interest Real-Time PCR Assays

pg 22-23

The other main factor that can reduce the priming efficiency, particularly at the limits of PCR detection, is primer dimer formation.

If the primers themselves have complimentary sequences then they can bind to each other leading to the production of short PCR products. Amplification of primer dimers robs the intended template of primers, enzyme and other reaction components thus reducing the PCR priming efficiency. Primers must be checked for cross homology so that primer interactions are reduced.

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Technical bulletin 06

Troubleshooting when using Intercalating Dyes (SYBR Green) ®

Introduction

In order to use intercalating dyes in real-time PCR successfully it is crucial to understand that data interpretation involves a combination of analysis of the amplification plots and the corresponding melt curves.

Melt curves Melt curves are a simple tool that essentially replace the running of post PCR gels that were required with standard old fashioned end point PCR. All real-time PCR machines are capable of running an automatic melt curve (sometimes called dissociation curve) as part of the real-time PCR protocol on the machine.

The PCR product is gradually heated from around 55°C to 95°C and fluorescent data is collected during the process. At the beginning of the programme, the DNA is double stranded, bound to SYBR® Green and therefore highly fluorescent. As the PCR product is heated up, eventually the melting temperature of the PCR product is reached and the two DNA strands separate releasing the SYBR® Green in to

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solution. This results in a sudden drop in fluorescence. Subsequently all of the PCR product is melted and the level of fluorescence plateaus off again.

Most real-time PCR machines take this data and present it more conveniently. Instead of plotting absolute fluorescence, the rate of change of fluorescence is plotted. Therefore, the sudden change in fluorescence seen at the melting temperature of the PCR product is visualised as a sharp peak against a relatively flat background where only a small change of fluorescence is occurring.

So in a perfect reaction a single distinct peak indicates that a single distinct PCR product has been amplified. It is analogous to seeing a distinct single band on a gel. The melting temperature of the PCR product can be predicted from the length of the amplicon and the proportion of G and C bases. So it is possible to check that the “correct” product has been amplified based upon the predicted melting temperature of the PCR product. Most real-time PCR amplicons will have a melting temperature of around 80-95°C when using our PrecisionPLUS Master Mix

If you see an imperfect melt curve you will need to interpret the result in order to understand the problem.

As a rule of thumb a melt peak with a Tm less than 80°C is most likely to be caused by primer dimers. Another clue is that primer dimers tend to melt over a wider temperature range resulting in a less sharp peak with a wider base. A primer dimer is formed where the forward and reverse primers bind to one another creating a polymerase binding site. After enough cycles of PCR the polymerase will eventually succeed in extending this product and amplifying it thus giving a false signal. This problem typically occurs when the target DNA is very rare, or even absent. In this scenario, with no authentic template to bind to, primers are much more likely to form dimers.

All of our primers are designed to have minimum propensity to form primer dimers, and in the presence of authentic template all of them will give specific amplification of the target of interest. However, even with the very best primers it is still sometimes possible to achieve a primer dimer signal after 33+ cycles of PCR when the

target of interest is absent. This is not necessarily a problem as long as you are capable of interpreting the melt curve correctly so that you ignore data from primer dimers and only work with data from authentic amplification plots.

If you are getting primer dimer signals that result in amplification plots with a Cq value of 30 or less then the primers are unsuitable and should be redesigned.

More often than not a melt curve with a melting temperature higher than the target of interest is caused by amplification on gDNA. An amplicon may be designed to span an intron to prevent detection of gDNA. However, if the intron is small it is sometimes possible to amplify across the intron creating a large PCR product. This is why it is best practice to DNase treat your RNA prior to the reverse transcription step as this problem is eliminated then and there.

Alternatively, a peak with an unexpected melting point may indicate that your primers are mis-priming and amplifying the wrong product.

Mis-priming on a more G/C rich template will give you an extra peak

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Technical bulletin 06

at a higher temperature just as mis-priming on a more A/T rich template will give you an extra peak at a lower melting temperature.

A signal in your no template control All good real-time PCR experiments have a no template control (NTC). This is where the DNA is replaced with water. A signal in the NTC indicates something is not perfect and should be investigated.

That said, it is not always a disaster.

NTC signals tend to come from one of two places:

Primer dimers As described above primer dimers are easy to spot on a melt curve. They are only a problem if they are interfering with the real signals from your real targets of interest. If a set of primers gives a good signal with a single melt peak when the target of interest is present but a primer dimer signal when it is absent this data is still interpretable. The limits of detection are defined by the point at which a primer dimer signal appears. As long as this is understood the data can still be scientifically valid.

If however, the primer dimer signal is present alongside your gene of interest signal - i.e. two peaks on your melt curve, then the data is uninterpretable and the primers will need to be redesigned.

PCR product contamination Real-time PCR generates billions of copies of your target of interest with every reaction. This PCR product is a massive potential contaminant for your future experiments. For this reason you should try never to open a PCR reaction after the PCR if you can help it.

Aerosols from opening a reaction plate can and will contaminate your laboratory. If you do need to open a PCR plate make sure it

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is in a different laboratory well away from where you set up your real-time PCR reactions. The danger with PCR product is that it is the perfect template for your real-time PCR primers. Therefore, if it ends up contaminating a future experiment it can lead to false positive results. This is why a NTC is so important. Amplification in the NTC of a product with a melting temperature the same as your target of interest shows that you have PCR product contamination in your laboratory/reagents. This is a very common problem but is not necessarily a disaster. Most laboratories will have a degree of persistent contamination with the targets of interest that are most frequently amplified in the lab. Again, the secret is to interpret your data correctly when contamination is present. If the contamination is so bad that the NTC Cq values are similar your “real” signal in your “real samples” then the data is not interpretable as you can’t be sure if the signal in your samples is biologically derived or as a result of contamination. However, if your real signals are greater than 6 cycles earlier than your NTC signals then you can be confident that your data is reliable and is not affected by this small amount of PCR product contamination.

If significant contamination has been identified you need to get rid of it quickly before repeating work. You can spend a great deal of time performing exclusion experiments to identify the precise cause, but PCR contamination is fiendishly hard to track down as the number of potential entry points is very high. It is usually much quicker to discard all currently reagents and primers for that target and decontaminate the lab. Pay particular attention to door handles, buttons, pipettes and surfaces. Start afresh by running a NTC control with no positive samples included so that you can be absolutely sure your reagents are now clean. This approach may initially seem wasteful but it can save a lot of human recourses and machine time.

Limit of Detections Intercalating dyes (e.g. SYBR® Green) work well when your target of interest is abundant. However, they run into problems when the target of interest is rare. Primer dimers and non specific binding events become much more common when the target gene is rare which makes using intercalating dyes problematic. Thus as a general rule of thumb the limits of detection when using an intercalating dye sit at around cycle number 30. If your target of interest takes greater than 30 cycles to cross the cycle threshold line then we would recommend switching to a probe based detection chemistry such as a hydrolysis (TaqMan®) probe.

Beyond this rule of thumb you can define your own limits of detection for your particular experiment by identifying the number of cycles of PCR where your signal becomes unreliable i.e. where primer dimers are present in your melt curves and/or when your NTC signals are less than 6 cycles later than ‘real’ signals.

Related products Custom Gene of Interest Real-Time PCR Assays qbase+ PrecisionPLUS Master Mix with SYBR Green

pg 22-23 pg 18-19 pg 28-29

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Technical bulletin 07

Normalisation Why Normalise?

During the preparation of cDNA for real-time PCR analysis there is significant potential for small errors to accumulate. For example, differences in sample size, RNA extraction efficiency, pipetting accuracy and reverse transcription efficiency will all add variability to your samples. Thus, if you are going to perform relative gene expression between samples, it becomes essential to answer the question; “how much cDNA is really present in these samples?”

Strategies to Consider When comparing one cDNA sample to another researchers have employed a number of strategies to try and “normalise” their data; •

Standardising sample size. Its good practice to try and use

similar sized samples (e.g. a similar number of cultured cells or similar sized biopsy), however this is insufficient as a normalisation strategy because it does not account for the cumulative errors that can occur in cDNA preparation • Normalising to genomic DNA. This strategy can be effective but is impractical because most RNA preparation protocols deliberately eliminate the presence of DNA • Normalising to total RNA. RNA quantification using a Bioanalyzer (Agilent) is a useful step. The analysis provide useful information about the quality of your RNA but again does not account for the cumulative errors that can occur in cDNA preparation •

Normalising to a reference (housekeeping) gene.

Normalising to a stably expressed reference gene that is representative of the cDNA concentration in a sample is the most

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commonly used normalisation approach. The reference gene is subject to the same errors in cDNA preparation as the gene of interest so makes an excellent normalising control. However, careful and strategic selection of the most stably expressed reference gene is essential. Random selection of a reference gene can add large unpredictable error to your analysis

Reference (housekeeping) Gene Selection Normalising real-time PCR data to the expression of a reference (housekeeping) gene is an excellent and practical strategy. However, reference genes are not stably expressed in all scenarios. Using the wrong reference gene for normalisation will make your data scientifically invalid. It is essential therefore to establish which reference genes are the most stably expressed in your particular experimental model. Your model is unlikely to be identical to anyone else’s so it is important to do this step for yourself.

Quick, Simple Reference Gene Selection with geNorm™ geNorm is a piece of software designed to establish the most stably expressed reference genes for any particular model. Following measurement of a number of candidate reference genes in 10 or so samples by real-time PCR the user inputs the data into geNorm. The software then carries out analysis of the relative ratio of expression of each reference gene. Thus the candidate reference genes are ranked in order of stability.

The software also provides useful information about the optimal number of reference genes that need to be averaged in order to

achieve the very best normalisation strategy. The geNorm software is a proven application and the original article is one of the most viewed articles on BioMed Central of all time, with over 2000 citations in other peer reviewed articles.

conditions are represented fairly. e.g. treated and untreated samples.

You need equal numbers of each. geNorm treats all samples the same and does not know which are treated and which are untreated so it is up to you as the user to represent them both equally so that geNorm can identify the genes most stably expressed across the experiment.

Q: How many samples should I use in my geNorm analysis?

A: A minimum of 8, but the more you run the better and more meaningful the data. 20 would be ideal.

Related products geNorm kits Reference gene assays

pg 14-15 pg 20-21

The geNormâ&#x201E;˘ Kit In collaboration with Biogazelle (Belgium), Primerdesign can provide geNorm kits for anyone working with human, rat or mouse samples plus a huge range of other species. The kit comprises 6 or 12 high quality reference gene real-time PCR assays and access to the latest geNorm software. The geNorm kit provides the simplest, quickest and most affordable route to establishing the best normalisation strategy for your real-time PCR research.

FAQâ&#x20AC;&#x2122;s Q: Which samples should I use in my geNorm analysis?

A: The most important thing to ensure is that all of the experimental

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Technical bulletin 08

Standard Curves – Measuring Primer Efficiency What is a Standard Curve?

A standard curve is a tool used in real-time PCR to measure the efficiency with which a given set of primers amplifies the target gene; the amplicon. In an ideal experiment a set of primers will work with 100% efficiency. This means that with every cycle of PCR the amount of amplicon present is multiplied by 2. If a primer set is working with less than 100% efficiency then the amount of amplicon is multiplied by less than 2 with each cycle of PCR. To perform a standard curve reaction the user takes their template DNA and performs a serial dilution. Typical this will be a 10 fold serial dilution over 6 points.

Then a graph of dilution vs Cq value is plotted:

Point 1: neat DNA Point 2: 1:10 dilution Point 3: 1:100 dilution Point 4: 1:1000 dilution Point 5: 1:10000 dilution Point 6: 1:100000 dilution The user then uses 5µl of this as the template for their real-time PCR. This results in 6 Cq values (sometimes known as Ct values).

The slope of this graph can then be converted into a percentage efficiency value. Most real-time PCR machine software packages do this automatically. But for simplicity’s sake it is easy to remember that a slope of -3.3 indicates an efficiency of 100%. If the slope is less than this then the priming efficiency is less than 100% (e.g.

Slope of -4.2 = efficiency of 73%)

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A good set of primers will report an efficiency between 90-110%, so you want to see a slope of between -3.1 and -3.6 in an ideal experiment.

The Problem with the Standard Curve Method

be perfectly good. Similarly, if the template contains impurities (proteins, lipids etc) this can result in a very different level of efficiency being reported. These impurities act as PCR inhibitors.

The standard curve is the best method that we have available to measure priming efficiency. However, it can be problematic. The reason for this is that the efficiency that a standard curve reports is influenced by more than just the priming efficiency. This leads to much frustration as different primer sets can report wildly different efficiencies on different days on different templates. There are two other major factors that alter the result obtained from a standard curve. It is important to understand both:

2. The abundance of the target gene can also hugely alter the result achieved from a standard curve experiment. When the target of interest is rare it is impossible to get a sufficient number of good data points as the dilution leads to the assay reaching the limits of detection. Thus if the target gene is abundant it tends to appear that a primer set is performing with high efficiency where as the same set of primers will report very low priming efficiency when using template with a low level of target gene present.

1. The template itself can massively alter the apparent result from a standard curve experiment. If the template in use is less than perfect, cDNA from degraded RNA for example, then this can result in a poor quality standard curve. The result will indicate the primers are performing with poor efficiency when in fact the primers may

Because the results obtained from a standard curve are so variable dependent on these additional factors, it makes it a somewhat scientifically flawed way to discover the true priming efficiency of a set of primers. If you have performed the standard curve method yourself using gDNA or cDNA you will no doubt have noticed how difficult it is to obtain consistent results from your standard curves.

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Technical bulletin 08

A solution to the Problem

these can massively alter the reported efficiency.

The solution to this problem is to standardise the template that is used. Biologically derived gDNA or cDNA are by their very nature, very variable. Instead it is best practice to use a copy of the amplicon cloned in to a vector. This provides a clean and stable template from which to create a standard curve. Such a tool will give reliably reproducible data day after day and most importantly will help you to very simply understand the real efficiency with which your primers are working. We can provide a copy number standard at 2 x 105 copies per microlitre if you require.

Q. What are the acceptable limits of priming efficiency to work within?

FAQs

A. First of all be certain that your primers really are outside of this

Q. M y standard curve indicates that my primers are priming with an efficiency of greater than 100%. How is this possible?

A. A priming efficiency of greater than 100% is of course beyond the theoretic limits of amplification since a PCR reaction can only double the amount of product in each PCR cycles and no more.

However, a reported priming efficiency of greater than 100% can be obtained from a standard curve. This results from a flattening of the standard curve gradient. In other words the amplification plots have been compressed together. When using SYBRÂŽ Green as the detecting chemistry, compression of the standard curve at late PCR cycles is commonly due to primer dimer formation, so make sure you check the melt curves before making a final judgement. It is always a good idea to remove outlying points as

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A. I deally you want a reported efficiency of 90-110%. This indicates that your primers are priming with an efficiency close to 100%.

This makes them suitable for the delta Cq method of data analysis. Life is simple in this instance.

Q. What do I do if my priming efficiency is outside of the range 90-110%? range. Consider all the things discussed in this bulletin. Then if your primers really do fall outside of this range then you have 2 options. You can try and correct for this poor efficiency within your calculations - the Pfaffl method - (this method should be used with caution as without a thorough expert understanding it can actually multiply errors rather than reducing them). Or you can throw out the primers and start again. We believe that the second option is the preferable one. It is more accurate to work with good quality primers than to try and correct for poor primers using mathematics.

Related products qbase+ pg 18-19 Custom designed positive control, for more information please contact enquiry@primerdesign.co.uk

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Technical bulletin 09

The Delta Cq Method – Relative Quantification

Introduction

In the vast majority of experiments the correct and most useful way to analyse gene expression data is by relative quantification. i.e. to asses how gene expression levels change between one subject group relative to another. This is different to absolute quantification but far simpler to execute and often far more accurate in its conclusions. The best and simplest method to calculate relative quantification is using the so called delta Cq method (also called the delta Ct method in the literature).

A Simple Principle When a PCR reaction is working well, with 100% efficiency, then every cycle of PCR doubles the amount of target DNA in the tube.

Based on this principle it is correct to conclude that if a PCR reaction reaches ‘take off point’ one cycle earlier than another PCR reaction then the first reaction contained twice as much target DNA as the second at the beginning of the reaction. It is this simple principle that underlies how real-time PCR can be used to produce accurate quantitative data.

One cycle earlier = twice the amount of target DNA present in the tube at the beginning of the reaction.

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Expressing this Mathematically To express this mathematically is simple. The difference between two samples is known as the delta Cq value (Delta is the Greek letter D and stands for ‘Difference’). So to compare two samples you simply subtract the Cq value of one sample away from the other to find the difference between the two.

Then you use the ‘delta Cq’ formula to convert this number to a relative ‘fold change’ in gene expression.

Relative level of expression = 2(delta Cq) So in this example 21 = 2 fold greater expression in sample A than sample B.

of PCR results in 2 fold increase in PCR product. If a PCR is less than 100% efficient then this is not true and the delta Cq formula is not appropriate to use. Pfaffl suggests in this instance that the formula be adjusted according to the real efficiency of the reaction in question. e.g. If the PCR is only 80% efficient each cycle of PCR results in a 1.8 fold increase in PCR product so the correct formula to use is 1.8delta Cq This technique is appropriate to use if your are forced into using an inherently inefficient set of primers. It relies on a very accurate analysis of priming efficiency to make it reliable, as any inaccuracy in calculating priming efficiency will result in errors being multiplied in downstream analysis. Because of this huge potential for error we recommend that if your primers are less than 90% efficient it is better practice to discard the primers and design new ones rather than to try and fix poor primers with mathematical corrections.

In this example the delta Cq is 11 (29-18=11) 211 = 2,048 fold greater expression in sample A than sample B

Any primers with a priming efficiency between 90% and 110% are suitable for use with the traditional delta Cq method, and we will guarantee a priming efficiency of greater than 90% for any primers that we design for you, thus making them very simple to work with.

The Pfaffl Method A very well cited variation of the delta Cq method is the so called Pfaffl method. Named after the esteemed author of the publication that described the approach (A new mathematical model for relative quantification in real-time RT–PCR. Michael W. Pfaffl, Nucleic Acids Res. 2001 May 1; 29(9): e45.)

The delta Cq formula 2delta Cq is based around the fact that every cycle

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Technical bulletin 10

Standard Curves – Measuring Absolute Copy Number Why Measure Absolute Copy Number?

Standard curves can be used in a real-time PCR experiment to quantify precisely the number of copies of a target DNA molecule present in a given sample*. In a lot of experiments this is an unnecessary thing to do.

It is important to ask yourself if there is any scientific benefit to know the precise number of copies of a target present. Does it have any biological meaning in your experimental model to know that, for example, there are 1000 copies per microlitre of your target present. More often than not it is more relevant to measure the fold change in expression of a target in a treated group vs a control group, or a healthy sample vs. diseased sample. In this case it’s more appropriate to use a technique such as the delta Cq method. However there are circumstances where knowing the precise number of copies of target present in a sample is crucial.

For example when using real-time PCR as a diagnostic tool to measure HIV viral load in a patient’s blood stream, as knowing the precise level of target present has a real clinical relevance as it effects the dose of anti retroviral drug to be administered.

The user takes their template DNA and performs a serial dilution.

Typically this will be a 10 fold serial dilution over 6 points.

Point 1: 2 x 105 copies per microlitre Point 2: 2 x 104 copies per microlitre Point 3: 2 x 103 copies per microlitre Point 4: 200 copies per microlitre Point 5: 20 copies per microlitre Point 6: 2 copies per microlitre The user then uses 5µl of this as the template for their real-time PCR. This results in 6 Cq values (sometimes known as Ct values).

How to Perform a Standard Curve to Measure Absolute Copy Number To perform a standard curve reaction to measure absolute copy number you first need a sample that contains your target at a known concentration. The best way to achieve this is to use a Primerdesign positive control which is a synthetic version of your target amplicon cloned in to a vector. It is then carefully calibrated and supplied at a precise concentration of 2 x 105 copies per microlitre. Thus if you use 5 microlitres in a real-time PCR reaction it will contain 1 million copies.

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Then a graph of copy number vs Cq value is plotted:

E.g. Copies per microlitre of serum E.g. Copies per cell E.g. Copies per gram of tissue *n.b. Standard curves can also be used to measure the efficiency with which a set of primers is working (see page 68). The focus of this technical bulletin however is on the use of standard curves to measure absolute copy number.

Related products Then the user can compare the Cq value that is achieved in an unknown sample with the standard curve and very simply â&#x20AC;&#x153;read offâ&#x20AC;? the number of target molecules present in the sample.

qbase+ pg 18-19 Custom designed positive control, for more information please contact enquiry@primerdesign.co.uk

Then the data can be expressed as a concentration relative to the original sample.

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Technical bulletin 11

The MIQE Guidelines Introduction During the past decade, several high-profile cases of faulty research have been linked to inconsistent real-time PCR techniques and experiments. In April 2009, Professor Stephen Bustin and an international team of nine scientists, joined forces and developed a set of guidelines for the publishing qPCR results. The resulting ‘MIQE guidelines’ outline the minimum information required to publish quantitative real-time PCR data with scientific integrity.

If you want to do real-time PCR properly, to produce scientifically relevant results and to get your data published then this is the one paper you should read:

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Clin Chem. 2009 Apr;55(4):611-22.

Primerdesign is cited in the MIQE guidelines because we share the same philosophy on primer sequences as the authors. We have always provided the primer sequences with our OnDemand Kits as we believe that this is crucial to perform research with integrity.

We have included a summary of the MIQE guidelines herein to act as a convenient guide for you. Don’t be daunted by the list. 90% of the

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items are common sense and you are probably already doing them.

However, there may be a few things in there that will make a real improvement to the quality of your data.

We strive to make all of our products compliant with the MIQE guidelines and will always be on hand to guide and advise you in producing real-time PCR data of the highest quality.

MIQE Checklist Key a All essential information (E) must be submitted with the manuscript. Desirable information (D) should be submitted if available. If primers are from RTPrimer DB, information on qPCR target, oligonucleotides, protocols, and validation is available from that source. b FFPE, formalin-fixed, paraffin-embedded; RIN, RNA integrity number; RQI, RNA quality indicator; GSP, gene-specific priming; dNTP, deoxynucleoside triphosphate. c Assessing the absence of DNA with ano–reverse transcription assay is essential when first extracting RNA. Once the sample has been validated as DNA free, inclusion of ano–reverse transcription control is desirable but no longer essential. d Disclosure of the probe sequence is highly desirable and strongly encouraged; however, because not all vendors of commercial predesigned assays provide this information, it cannot be an essential requirement. Use of such assays is discouraged.

MINIMUM INFORMATION FOR PUBLICATION OF QUANTITATIVE REAL-TIME PCR EXPERIMENTS MIQE CHECKLIST FOR AUTHORS, REVIEWERS, AND EDITORS.a ITEM TO CHECK

IMPORTANCE

EXPERIMENTAL DESIGN Definition of experimental and control groups

E

Number within each group

E

Assay carried out by the core or investigator’s laboratory?

D

Acknowledgment of authors’ contributions

D

SAMPLE Description

E

Volume/mass of sample processed

D

Micro dissection or macrodissection

E

Processing procedure

E

If frozen, how and how quickly?

E

If fixed, with what and how quickly?

E

Sample storage conditions and duration (especially for FFPEb samples)

E

NUCLEIC ACID EXTRACTION Procedure and/or instrumentation

E

Name of kit and details of any modifications

E

Source of additional reagents used

D

Details of DNase or RNase treatment

E

Contamination assessment (DNA or RNA)

E

Nucleic acid quantification

E

Instrument and method

E

Purity (A260/A280)

D

Yield

D

RNA integrity:method/instrument

E

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Technical bulletin 11

RIN/RQI or Cq of 3’and 5’ transcripts

E

Electrophoresis traces

D

Inhibition testing (Cq dilutions, spike, or other)

E

REVERSE TRANSCRIPTION Complete reaction conditions

E

Amount of RNA and reaction volume

E

Priming oligonucleotide (if using GSP) and concentration

E

Reverse transcriptase and concentration

E

Temperature and time

E

Manufacturer of reagents and catalogue numbers

D

Cqs with and without reverse transcription

Dc

Storage conditions of cDNA

D

qPCR TARGET INFORMATION Gene symbol

E

Sequence accession number

E

Location of amplicon

D

Amplicon length

E

Insilico specificity screen (BLAST, and soon)

E

Pseudogenes, retropseudogenes or other homologs?

D

Sequence alignment

D

Secondary structure analysis of amplicon

D

Location of each primer by exon or intron (if applicable)

E

What splice variants are targeted?

E

qPCR OLIGONUCLEOTIDES Primer sequences

E

RTPrimer D B identification number

D

Probe sequences

Dd

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Location and identity of any modifications

E

Manufacturer of oligonucleotides

D

Purification method

D

qPCR PROTOCOL Complete reaction conditions

E

Reaction volume and amount of cDNA/DNA

E

2

Primer, (probe), Mg +, and dNTP concentrations

E

Polymerase identity and concentration

E

Buffer/kit identity and manufacturer

E

Exact chemical composition of the buffer

D

Additives (SYBR Green I, DMSO and so forth)

E

Manufacturer of plates/tubes and catalog number

D

Complete thermocycling parameters

E

Reaction set up (manual/robotic)

D

Manufacturer of qPCR instrument

E

qPCR VALIDATION Evidence of optimisation (from gradients)

D

Specificity (gel, sequence, melt or digest)

E

For SYBR Green I, Cq of the NTC

E

Calibration curves with slope and y intercept

E

PCR efficiency calculated from slope

E

CIs for PCR efficiency or SE

D

r2 of calibration curve

E

Linear dynamic range

E

Cq variation at LOD

E

CIs throughout range

D

Evidence for LOD

E

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Technical bulletin 11

If multiplex, efficiency and LOD of each assay

E

DATA ANALYSIS qPCR analysis program (source, version)

E

Method of Cq determination

E

Outlier identification and disposition

E

Results for NTCs

E

Justification of number and choice of reference genes

E

Description of normalisation method

E

Number and concordance of biological replicates

D

Number and stage (reverse transcription or qPCR) of technical replicates

E

Repeatability (intra assay variation)

E

Reproducibility (interassay variation, CV)

D

Power analysis

D

Statistical methods for results significance

E

Software (source, version)

E

Cq or raw data submission with RDML

D

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Appendix

Our catalogue of reference (housekeeping) genes includes (but not limited to): Human (Homo sapiens): • Actin, beta (ACTB) • Glyceraldehydes phosphate dehydrogenase (GAPDH) • Ubiquitin C (UBC) • Beta-2-microglobulin (B2M) • Phospholipase A2 (YWHAZ) • Ribosomal protein L13a (RPL13A) • 18S Ribosomal RNA (18S) • Cytochrome c-1 (CYC1) • Eukaryotic translation initiation factor 4A (EIF4A2) • Homo sapiens succinate dehydrogenase (SDHA) • DNA topoisomerase I (TOP1) • ATP synthase, (ATP5B) • Excision repair cross-complementing rodent repair deficiency (ERCC6) • Ubiquitin-conjugating enzyme E2D 2 (UBE2D2) • Ubiquitination factor E4A (UBE4A) • Ecto-NOX disulfide-thiol exchanger 2 (ENOX2) • PR domain containing 4 (PRDM4) • Selenocysteine lyase (SCLY) • tRNA-yW synthesizing protein 1 homolog (S. cerevisiae) (TYW1) • Ring finger protein 20 (RNF20) • Chromosome 14 open reading frame 133 (C14orf133) Mouse (Mus musculus): • Actin, beta, cytoplasmic (ACTB) • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) • Ubiquitin C (UBC) • Beta-2 microglobulin (B2M) • Phospholipase A2 (YWHAZ) • Ribosomal protein L13a (RPL13A) • Calnexin (CANX) • Cytochrome c-1 (CYC1) • Succinate dehydrogenase complex, subunit A (SDHA) • 18S Ribosomal RNA (18S)

• Eukaryotic translation initiation factor 4A2 (EIF4A2) • ATP synthase subunit (ATP5B) • Adaptor-related protein complex 3 (Ap3d1) • Casein kinase 2, alpha prime polypeptide (Csnk2a2) • Cell division cycle 40 homolog (Cdc40) • F-box and WD-40 domain protein 2 (Fbxw2) • F-box protein 38 (Fbxo38) • HIV TAT specific factor 1 (Htatsf1) • MON2 homolog (Mon2) • PAK1 interacting protein 1 (Pak1ip1) • Zinc finger protein 91 (Zfp91) Rat (Rattus Norvegicus): • 18S Ribosomal RNA (18S) • ATP synthase, (ATP5B) • Topoisomerase I (TOP1) • Malate dehydrogenase 1 (MDH1) • Cytochrome c-1 (CYC1) • Calnexin (CANX) • Ribosomal protein L13 (RPL13) • Tyrosine 3-monooxygenase (YWHAZ) • Beta-2 microglobulin (B2M) • Ubiquitin C (UBC) • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) • Actin, beta (ACTB) • Nucleoporin like 2 (Nupl2) • Similar to XPA binding protein 1 (LOC688393) • Staufen RNA binding protein homolog 1 (Stau1) • F-box and WD repeat domain containing 4 (Fbxw4), mRNA • CCCH-type with G patch domain (Zgpat) • Integrin-linked kinase-associated serine/threonine phosphatase 2C (Ilkap) • FtsJ methyltransferase domain containing 2 (Ftsjd2) • Tomm22 nuclear gene encoding mitochondrial protein (Tomm22)

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Notes

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Notes

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ampliSOLVE®, genesig®, oasig® and the Primerdesign logo are all trademarks of Primerdesign Ltd. The PCR process is covered by US Patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG. SYBR® Green is a registered trade mark of Molecular Probes Inc. ABI, ABI PRISM® GeneAmp® and iCycler™ is a registered trademark of Bio-Rad Laboratories, Rotor-Gene is a trademark of QIAGEN. LightCycler™ is a registered trademark of the Idaho Technology Inc. GeneAmp®, TaqMan® and AmpliTaqGold® are registered trademarks of Roche Molecular Systems, Inc. The purchase of the Primerdesign™ reagents cannot be construed as an authorisation or implicit licence to practice PCR under any patents held

by Hoffmann-LaRoche Inc.

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We are always delighted to help with any aspect of your real-time PCR research. Please feel free to contact us for free advice or technical support.

Primerdesign Ltd York House School Lane Chandlers Ford United Kingdom SO53 4DG Telephone: +44 (0)23 8074 8830 Fax: +44 (0)870 836 2155 Orders: orders@primerdesign.co.uk Enquiries: enquiry@primerdesign.co.uk Technical support: support@primerdesign.co.uk facebook.com/realtimePCR twitter.com/PrimerdesignLtd linkedin.com/company/Primerdesign youtube.com/Primerdesign Brochure version MM004 Issue 04

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