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Application note Using siRNA libraries

Assessing siRNA efficiency by cell morphology & proliferation studies It has long been an essential part of biology to investigate the effect of every gene in the genome on a gene-to-gene basis. In 1998, Fire et al. [1] published a paper describing how short double-stranded RNA (dsRNA) molecules could silence specific genes in C. elegans. Furthermore, it did not require an immense amount of the dsRNA molecules to achieve complete gene silencing. They called the phenomenon RNA interference (RNAi) and the discovery gave Fire and Mellow the Nobel Prize in Physiology or Medicine 2006. When dsRNA is inserted into a cell it is broken down into shorter dsRNA strands called small interfering RNA (siRNA) or micro RNA (miRNA). These strands can then be separated and mounted on an RNA-induced silencing complex (RISC) after which they proceed to silence the mRNA of the targeted gene [2]. Since the initial discovery, siRNA and miRNA have been found in most living organisms.

Endpoint

The effect of siRNA is often evaluated through proliferation assays, gene expression and mRNA assays. The HoloMonitorTM M3 can be used to assess changes in cell proliferation that are induced by siRNA treatment. The HoloMonitorTM M3 is an excellent complementary instrument that increases the information for each experiment tremendously as cell proliferation, cell morphology and other parameters can be measured easily and non-destructively.

Application

To analyze the siRNA effects on proliferation and morphology, samples are placed on the objective table of

the HoloMonitorTM M3. The study can then be performed either as a timelapse study, in which case the cells should be put on a heating stage or in a micro-incubator, or as separate time-points after treatment, in which case no special equipment is necessary. Images are captured automatically or manually, as set by the operator. After completion of the image capture, the images are processed and reconstructed as previously described (MĂślder et al., 2008). Reconstructed phase images (Fig. 1) provide the starting point for further image analysis. Cell number as well as confluence can be determined. Further parameters like cell shape, size and thickness can be measured. Using the HoloMonitorTM M3 it is possible to capture images of cells grown in almost all standard cell culture containers. The use of HoloMonitorTM M3 does not hinder or affect the possibility to use other methods as end-points. HoloMonitorTM M3 will therefore allow a user to extract more data from each experiment for a negligible amount of time and with no extra material required. There is no theoretical limit to which or how many time-points the HoloMonitorTM M3 can be used.

For these parameters an effect of siRNA is clear already after 1 day. Cells treated with siRNA are smaller and thicker than control cells.

A

B

Confluence (%)

Background

C

D

Results

The effect of siRNA as measured by cell number is quite clear after 2 days. The siRNA inhibits cell growth (Fig. 2A). The confluence curves follow the cell number curves (Fig. 2B), and may preferably be used for cells that tend to clump together. These proliferation parameters indicate differences one day later than morphological parameters such as cell thickness (Fig. 2C) and cell area (Fig.2D).

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Figure 1. The effect of siRNA treatment on L929 cells (circle) compared to control (triangle) as measured using the HoloMonitor™ M3. A represents cell number per mm2, B represents the area of the image that is covered with cells, i.e. the confluence, C represents the mean area of the individual cells and D represent the mean thickness of the individual cells.


Application note

Discussion

While the HoloMonitorTM M3 cannot replace any method that measures gene expression, it is an excellent complementary tool to analyze the net effects any change in gene expression may have on cell morphology and proliferation. Morphology changes are often apparent before proliferation changes, and HoloMonitorTM M3 offers the possibility of statistically valid morphology studies. During in-house experiments it has been shown that the HoloMonitorTM M3 can pick up changes in morphological data well before a human

observer would notice it. The HoloMonitorTM M3 allows users to continuously and non-invasively study the morphology and proliferation of their in vitro cell cultures without compromising the culture. This makes it possible to use the cell cultures for other experiments after finishing the proliferation assay with HoloMonitorTM M3. As has been shown, the effect of siRNA often takes time to show. References [1] Fire, A., Xu, SQ., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. Potent and specific genetic interference by

Figure 2. The thickness of control and siRNA-treated cells. When using the HoloMonitor™ M3 for siRNA assessing of gene functions, the results include not only cell proliferation and confluence, but also quantifiable morphological parameters such as cell thickness, area and shape. The changes in morphological parameters most often precede proliferation changes.

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double-stranded RNA in Caenorhabditis elegans. Nature 391, p. 806-811, 1998. [2] Montgomery, M.K., Xu, SQ., Fire, A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Procedings of the National Academy of Sciences of the United States of America 95 (26), p. 15502-15507, 1998. [3] Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. Duplexes of 21-nucleotid RNAs mediate RNA interference in cultures mammalian cells. Nature 411, p. 494-498, 2001. [4] Gartel, A.L., Kandel, E.S. RNA interference in cancer. Biomolecular Engineering 23, p. 17-34, 2006.


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