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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
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