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Measuring Gene Silencing Effects by RT-PCR Without RNA Isolation

Perform RT-PCR Directly in Cell Lysates Using Cells-to-cDNA II

Quantitative or real time RT-PCR is one of the most commonly used techniques for quantitating changes in gene expression across samples. When working with cell or tissue samples for subsequent use in RT-PCR you must extract RNA, DNase I treat the sample to eliminate contaminating genomic DNA, and then inactivate the DNase I with a Proteinase K treatment and/or a phenol extraction step. If only small numbers of cells (or limited amounts of tissue) are available, there is a risk of losing some of the RNA sample during these steps. Here we describe how Ambion's Cells-to-cDNA II Kit can be used with siRNA transfected cells to measure gene silencing directly in cell lysates, without the need to extract RNA.

Cells-to-cDNA II: Reverse Transcription without RNA Isolation
The RNA isolation step is usually the most laborious and time-consuming part of any RNA analysis protocol, especially when multiple samples need to be analyzed. Ambion's Cells-to-cDNA II Kit (patent pending) offers a significantly more convenient and streamlined procedure to assess gene expression changes in cell samples via RT-PCR. The Cells-to-cDNA II Kit yields cDNA from cultured mammalian cells in less than 2 hours without RNA isolation. The procedure is simple. Cells are lysed in the Cell Lysis Buffer included in the kit, and DNase I treated. cDNA synthesis is then performed directly with this lysate . A Cells-to-cDNA II-96 Automated Kit is also available for high-throughput cDNA synthesis on robotic platforms. In addition to offering the advantages of high-throughput analyses, the automated platform shows clear reproducible measurements of changes in gene expression. This is especially critical for obtaining meaningful data when measuring changes in gene expression over large sample numbers subjected to different kinds of treatment or stimuli.

Measuring the siRNA Effect
To demonstrate the use of the Cells-to-cDNA II-96 Automated Kit for measuring siRNA induced knockdown of gene expression, scientists at Ambion performed a study using cultured HeLa cells transfected with chemically synthesized siRNAs targeting cdc-2, GAPDH, and survivin. Briefly, 3000 HeLa cells were plated into wells of a 96-well culture plate and allowed to adhere overnight at 37C. The cells were then transfected with 100 nM of each siRNA or a scrambled siRNA control, and incubated at 37C for 48 hours. The cells were then washed, lysed and DNase treated according to the Cells-to-cDNA II-96 protocol developed for the Biomek 2000 Multichannel Liquid Handling System (Beckman-Coulter). 5 l of lysate was used in a 25 l one-step, real-time RT-PCR with TaqMan probes. A probe for 18S rRNA was used to normalize the data from each experiment. Experiments were performed in replicates of eight. As can be seen from Figure 1, gene silencing is readily detectable by real time RT-PCR and the data generated using the Cells-to-cDNA II-96 Kit is highly reproducible.

Figure 1. Use of Cells-to-cDN A II-96 Automated Kit and qRT-PCR to Measure Gene Silencing. HeLa cells were transfected with scrambled control (red) and gene specific siRNAs to cdc-2, GAPDH and survivin (blue). 18S rRNA was used as a reference standard for quantitation. The 18S rRNA signal was measured for cells transfected with the scrambled siRNA control (green) and the gene specific siRNAs (black, running over green).

A Complete Kit for Cell Lysis and cDNA Synthesis
Real time RT-PCR with the Cells-to-cDNA II Kit offers a versatile method to measure the effects of siRNA mediated gene silencing at the RNA level. Because the RNA isolation step is eliminated, multiple small samples can be readily analyzed. The kit can be used with transfected or treated cultured cells, and is also compatible with LCM and FACS sorted samples. The Cells-to-cDNA II Kit contains all the necessary reagents for 40 or 100 reverse transcription reactions, including M-MLV RT and oligo(dT)18 or random decamer primers. From 1 to 12,500 cells can typically be used in each Cells-to-cDNA RT-PCR reaction either in individual tubes or in a 96 well format. The version of the kit used with automated liquid handling systems makes it an ideal technology to facilitate large scale RNAi experiments.

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Ordering Information
Cat# Product Name Size 1722 Cells-to-cDNA II Kit 40 rxns 1723 Cells-to-cDNA II Kit 100 rxns 2050 SuperTaq Polymerase (Cloned) 5 U/l 50 U 8723 Cells-to-cDNA II Cell Lysis Buffer 5 x 10 ml


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