Real-time, quantitative RT-PCR (qRT-PCR) is the most sensitive way to detect and quantitate mRNA and is often used to validate techniques that monitor changes in gene expression, including array analyses and RNA interference experiments. The Cells-to-Signal Kit (patent pending) is a fast way to process samples for qRT-PCR from as few as three cells. Lysates made with the Cells-to-Signal Kit are compatible with qRT-PCR using either TaqMan or SYBR Green detection.
RNA interference (RNAi) has become an important tool for understanding gene function. Researchers increasingly rely on qRT-PCR to detect and quantitate mRNA levels to confirm knockdown of gene expression by an siRNA. Ambion's Cells-to-Signal Kit greatly simplifies this procedure through bypassing RNA isolation and DNA removal (Figure 1). Cell lysates produced with the Cells-to-Signal Kit can be used directly in qRT-PCR when PCR primers are designed to span exon-exon boundaries. Here, we show that Cells-to-Signal lysates are compatible with TaqMan probes and SYBR Green detection methods.
Figure 1. Cells-to-Signal
Procedure. After a ~5 min cell lysis
procedure, lysates can be used directly for RT-PCR, as long as primer
pairs span exon-exon boundaries. Minus reverse transcriptase control reactions
should also be included to monitor potential amplification of genomic
sequences. Sequence specificity of the TaqMan probes permits one-step