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 or two-step RT-PCR, while detection with SYBR Green, which binds to any double-stranded DNA molecule, requires a two-step RT-PCR to decrease nonspecific background.
qRT-PCR with TaqMan Probes
TaqMan Probes are short oligonucleotides that are homologous to an internal region of the PCR product and are labeled with a 5' fluorophore and 3' quencher. Quantification of PCR amplification relies on the 5' exonuclease activity of Taq polymerase to degrade the probe and separate the fluorophore from the quencher.
Cells-to-Signal lysates are ideal for use in one-step qRT-PCR using TaqMan probes. As shown in Figure 2, HeLa cells were transfected with 30 nM siRNA targeting GAPDH or 30 nM negative control siRNA. 48 hours post-transfection, the Cells-to-Signal Kit was used to prepare cell lysates for qRT-PCR using TaqMan Primer & Probe Sets. Relative to the negative control siRNA, GAPDH-specific siRNA knocked down GAPDH gene expression by more than 80%.
Figure 2. Measuring siRNA-induced Knockdown of Gene Expression with TaqMan Primer and Probe Sets. HeLa cells were plated in a 24 well plate (3 x 104 cells/well). Cells were transfected with 30 nM of either Silencer Negative Control #1 siRNA (Ambion) or Silencer GAPDH siRNA (Ambion). After 48 h, the cells were harvested and lysed with the Cells-to-Signal Kit (Ambion) (final volume = 500 l). 3 l of the cell lysate was used in one-step RT-PCR, using TaqMan Primer & Probe sets on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). GAPDH signals were normalized using 18S rRNA as an internal control. Refer to inset table for Ct values.
qRT-PCR with SYBR Green Detection
SYBR Green is an economical nucleic acid stain that binds to double-stranded DNA and can be used to detect PCR products on most real-time detection platforms. Although this method usually requires more optimization than techniques that use labeled probes, SYBR Green is compatible with Cells-to-Signal lysates and two-step qRT-PCR.
To ensure accurate analysis, the product dissociation curve should be evaluated after PCR. For example, HeLa cells were transfected with 30 nM siRNA targeting GAPDH or a negative control siRNA. 72 hours post-transfection, the Cells-to-Signal Kit was used to prepare cell lysates for qRT-PCR. As expected, the expression level of GAPDH was decreased by more than 80% relative to cells transfected with a negative control (Figure 3). To show that the relative fluorescence of SYBR Green in the amplification curve results from interactions with specific PCR products, the dissociation curve for each product was plotted (Figure 3, Inset). The defined peaks at the expected Tm are indicative of specific GAPDH and 18S rRNA amplification from the Cells-to-Signal lysates.
Figure 3. Measuring siRNA-induced Knockdown of Gene Expression with SYBR Green. HeLa cells were plated in a 96 well plate (8 x 103 cells/well). Cells were transfected with 30 nM of either Silencer Negative Control #1 siRNA (Ambion) or Silencer GAPDH siRNA (Ambion). After 72 h, the cells were harvested and lysed with the Cells-to-Signal Kit (Ambion) (final volume = 100 l). 3 l of the cell lysate was used in two-step RT-PCR, using SYBR Green on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). GAPDH signals were normalized using 18S rRNA as an internal control. These amplification curves show relative fluorescence and Ct values for 18S rRNA, and GAPDH from negative control siRNA-transfected cells and from GAPDH siRNA-transfected cells. For the four transfection replicates, percent expression knockdown of GAPDH was 80 4.3%. (Inset) The dissociation curves for each sample indicate that the desired target amplicons were the dominant products of the PCR.
Cell Lysis and cDNA Synthesis
Ambion's Cells-to-Signal Kit was developed specifically for quantitating gene expression in cells by real-time RT-PCR and is ideal for monitoring siRNA-induced knockdown. Cells-to-Signal lysates are compatible with one-step or two-step RT-PCR and both TaqMan probe and SYBR Green detection technologies. The Cells-to-Signal Kit contains reagents for cell lysis and reverse transcription, including M-MLV RT, oligo(dT)18 primers, and random decamer primers. Sample sizes up to 5 x 104 cells per reaction can be used. The kit also contains a control RNA for monitoring RT-PCR inhibition and complementary primers for optimizing reverse transcription efficiency.
Jennifer Ho, Kevin Kelnar, Quoc Hoang, Rich Jarvis Ambion, Inc.