- Quantitate gene expression in cells — without isolating RNA
- 5 minute cell lysis procedure
- qRT-PCR from as few as 3 cells
- Ideal for monitoring siRNA-induced knockdown
- Easily adapted to high throughput analysis
Real-time quantitative RT-PCR (qRT-PCR) is the most sensitive technique for mRNA detection and quantitation currently available. qRT-PCR is also the preferred method for validating array analysis results, RNA interference experiments, and other techniques that evaluate gene expression changes on a global scale. Ambion’s Cells-to-Signal Kit uses a chemical lysis method to create cell lysates in less than 5 minutes at room temperature (Figure 1). The cell lysate can then be used directly for RT-PCR, bypassing RNA isolation altogether. (The PCR primers must be designed to flank a large intron in the genomic DNA or span an exon-exon boundary in the cDNA. The intervening intron will then inhibit undesired amplification of genomic DNA in the cell lysate.) The Cells-to-Signal Kit is compatible with a wide variety of cell types and yields data that is highly reproducible. This kit is an improved version of Ambion's popular Cells-to-cDNA II Kit and has been optimized for high throughput analyses.
Compatible with TaqMan® Gene Expression Assays
Applied Biosystems pre-designed primers and probe sets for real-time PCR are some of the most commonly used tools for measuring changes in gene expression by qRT-PCR. The Cells-to-Signal technology is compatible with TaqMan Gene Expression Assays. Figures 2 and 3 show data generated in one-step real-time RT-PCR using TaqMan Gene Expression Assays for human GSK-3beta and VEG-F. In addition, the data also demonstrate that the Cells-to-Signal procedure is sensitive enough to enable target detection from as few as 3 cells (Figure 3).
NOTE: AmpliTaq® Gold DNA Polymerase (Applied Biosystems) is not compatible with the lysate or cDNA prepared using this kit. For optimal performance, use Ambion's SuperTaq™ DNA Polymerase.
Ideal for Measuring siRNA Knockdown
siRNA induced knockdown of gene expression is now one of the most widely used methods to determine gene function. Scientists at Ambion performed a study using cultured HeLa and MCF-7 cells transfected with 30 nM of chemically synthesized siRNA targeting GAPDH to demonstrate the effectiveness of the Cells-to-Signal Kit for measuring knockdown of gene expression (Figure 4). As expected, the expression levels of both genes were down-regulated by more than 80% compared to cells transfected with a scrambled control, while the expression of 18S rRNA was unchanged.
Easily Adapted for High Throughput Analyses
The quick and simple Cells-to-Signal protocol can be easily adapted to a 96 or 384 well format, thus enabling the simultaneous analyses of multiple cell samples. The protocol affords excellent reproducibility as seen in Figure 5, where VEG-F and GAPDH expression levels were determined in HeLa and MCF-7 cells grown in a 96 well plate.
Complete Kit
The Cells-to-Signal Kits contain reagents for 30 or 100 reverse transcription reactions, including M-MLV RT, oligo(dT)18 primers, and random decamer primers. Up to 5 x 104 cells per reaction can be used for the lysis step, and lysate can comprise up to 30% of the RT-PCR reaction. Ambion scientists have used the Cells-to-Signal Kit with such cell lines as HeLa, HeLa S3, MCF-7, K562, SKNAS and NHDF-neo, and we continue to test other cell types. The kit contains complementary primers and a positive control RNA for optimizing the procedure.
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