A)
B)
RNeasy Kit (Qiagen), replicates 5–8
1400 [nt]
10000 8000
28S -
- 4000
18S -
- 2000 - 1000 - 500 - 200 - 25 1
2
3
4
5
6
7
1600
Yield (µg of protein)
illustra triplePrep Kit (GE Healthcare), replicates 1–4
8
6000 4000
18S ribosomal RNA cytochrome P450 c-fos gene
2000
0
5
1000 800
- 4000
600
2D-DIGE - 2000 reference - 1000 method - 500
illustra triplePrep Kit
400
- 200
200
- 25
0
10 15 20 25 30 35 40 45 50 Cycle
Fig 3. Total RNA isolation from 10 mg of rat liver tissue using the illustra triplePrep Kit and RNeasy Mini Kits. (A) Analysis using Agilent Bioanalyzer revealed intact 18S and 28S ribosomal RNA bands indicating high quality RNA. (B) RT-qPCR results for amplified high-, medium-, and low-expressed 18S ribosomal RNA, cytochrome P450, and c-fos gene.
[nt]
1200
Innovations Forum
Nucleic acid purification
A)
2
3
4
Protein 6 7
5
8
B)
Total protein
Conclusions illustra triplePrep Kit provides rapid isolation of gDNA, RNA, and protein from single undivided samples. This kit enables researchers to correlate DNA, RNA, and protein data from the same sample and generate data related to gene expression, protein expression, and genomic analysis from precious limited samples such as biopsies, archived tissues, and tumors. Comparative analysis with DNeasy Blood and Tissue Kit, RNeasy Mini Kit, and the 2-D DIGE reference method showed higher yields of DNA, RNA, and total protein, respectively with equal or better quality enabling successful applications in PCR, RT-qPCR, sequencing, Western blotting, and LC-MS.
References 1. Henkel, C. et al. Changes of the hepatic proteome in murine models for toxically induced fibrogenesis and sclerosing. Proteomics 6, 6538–6548 (2006). 2. Aljanabi, S.M. and Martinez, I. Universal and rapid salt-extraction of high quality genomic DNA for PCR- based techniques Nucl. Acids Res. 25, 4692–4693 (1997). 3. Vogelstein, B. and Gillespie, D. Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615 (1979).
HeLa cells
C)
Rat liver
100 90 80
Relative abundance
Proteins were isolated from the second flowthrough after isolating gDNA and total RNA following the illustra triplePrep protocol. Yield was similar for the both illustra triplePrep Kit and 2D-DIGE reference method (Fig 4A). Western blotting using appropriate antibodies was used to detect β-actin or GAPDH proteins (Fig 4B). The peptides were separated by nanoRPC on Ettan™ MDLC coupled to a Finnigan LTQ™ Linear Ion Trap mass spectrometer (Thermo Fisher Scientific Inc.). The LC-MS data was evaluated using DeCyder™ MS Differential Analysis Software. Protein samples from the illustra triplePrep Kit extraction were also analyzed by LC-MS for peptide profiling and showed average representation of 3115 ± 176 peptides (Fig 4C).
70 60
39.04 30.96
20.78
40.48
50
44.47
28.05
40
46.21 34.89
30 20 50.78
55.06 59.7561.03 64.18
19.56
0
10.6515.0616.24
0
5
54.14
48.06
22.11 26.61
10
73.00
10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (min)
Fig 4. (A) Protein isolated from the illustra triplePrep Kit and 2-D DIGE reference method produced similar yields. (B) Western blotting analysis of proteins shows the presence of β-actin and GAPDH. (C) The base peak ion chromatogram of LC-MS shows a wide peptide distribution and signal intensity from protein isolated by illustra triplePrep Kit.
Ordering information Product
Code number
illustra triplePrep Kit, 50 preps
28-9425-44
2-D Quant Kit
80-6483-56
For more information on our range of protein and nucleic acid sample preparation products, visit www.gelifesciences.com/illustra
4. Sambrook, J. and Russell, D. W., Molecular Cloning: A Laboratory Manual, chapter 6, Cold Spring Harbor Laboratory Press, New York (2001).
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