Go from Cells in Culture to Real-Time PCR Data Even Faster
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The first step in gene expression analysis using RT-PCR is the recovery of pure RNA from experimental samples. Even the quickest and simplest techniques may require 30 minutes or more of hands-on time. TaqMan® Cells-to-CT technology enables reverse transcription of lysates directly from 10 to 105 cultured cells without isolating or purifying RNA, saving valuable time and effort. When combined with real-time PCR methods, the TaqMan Cells-to-CT kits generate data equivalent to that generated using purified RNA, and the technology shows distinct advantages at low cell input.
The new TaqMan Fast Cells-to-CT Kit combines the advantages of Cells-to-CT sample preparation with the reduced thermal cycling times offered by TaqMan Fast Universal Master Mix to further expedite gene expression analysis for researchers using cells in culture. Fast cycling conditions dramatically decrease the time to results relative to standard cycling.
The new TaqMan Fast Cells-to-CT Kit combines the advantages of Cells-to-CT sample preparation with the reduced thermal cycling times offered by TaqMan Fast Universal Master Mix to further expedite gene expression analysis for researchers using cells in culture. Fast cycling conditions dramatically decrease the time to results relative to standard cycling.
Fast 7-Minute Sample Prep
In 7 minutes your cell sample is lysed and treated concurrently with DNase (optional). All steps are performed at room temperature, and no centrifugation is needed. Cells can be lysed directly in 96- or 384-well cell culture plates for high-throughput applications. This simple, direct method reduces sample loss and the potential for error due to sample handling in multiple step procedures (Figure 1).
Sensitivity/Dynamic Range
Lysates from the TaqMan Fast Cells-to-CT Kit produce linear signal in real-time PCR across 5 logs of cellular input, from 10 to 105 cells, making it the ideal kit for the analysis of small or large cell samples.
Unlike competitor kits that limit sensitivity of detection because only 5% of the lysate can be used in the RT reaction, the TaqMan Fast Cells-to-CT Kit can accommodate 45% of the total RT reaction volume. Also 30% of the resulting cDNA can be added to the real-time PCR. Because a large amount of the lysate can be carried over into the subsequent enzymatic reactions, the TaqMan Fast Cells-to-CT Kit is highly sensitive. The TaqMan Fast Universal PCR Master Mix included in the kit is precise and accurate, enabling the detection of small quantities of target, including transcripts expressed at low levels.
A synthetic template was used to validate the performance of TaqMan Fast Universal Master Mix in the TaqMan Fast Cells-to-CT Kit. Real-time PCR sensitivity and dynamic range were evaluated by amplifying a 9-log range of synthetic template concentration in a background of cDNA prepared either from purified RNA or from a TaqMan Cells-to-CT lysate. The data show equivalent target detection over the entire range of input template amounts, indicating that there are no inhibitory or negative effects of the lysate on the sensitivity of the PCR (Figure 2).
Figure 2. TaqMan® Fast Cells-to-CT™ Lysates and Purified RNA Give Comparable Results in Real-Time PCR. A 50 µL RT reaction was performed that contained either 10 µL of TaqMan Cells-to-CT Kit lysate (10,000 NIH/3T3 cells in 50 µL original lysate) or equivalent amounts of purified RNA. A constant volume (20% total PCR volume) of the RT reactions was combined with the indicated amounts of diluted synthetic target for the Hs00430940_m1 assay and analyzed using the assay specific for that target. Results are virtually indistinguishable between reactions performed in a background of purified RNA vs. TaqMan Cells-to-CT Kit lysate.
Unlike competitor kits that limit sensitivity of detection because only 5% of the lysate can be used in the RT reaction, the TaqMan Fast Cells-to-CT Kit can accommodate 45% of the total RT reaction volume. Also 30% of the resulting cDNA can be added to the real-time PCR. Because a large amount of the lysate can be carried over into the subsequent enzymatic reactions, the TaqMan Fast Cells-to-CT Kit is highly sensitive. The TaqMan Fast Universal PCR Master Mix included in the kit is precise and accurate, enabling the detection of small quantities of target, including transcripts expressed at low levels.
A synthetic template was used to validate the performance of TaqMan Fast Universal Master Mix in the TaqMan Fast Cells-to-CT Kit. Real-time PCR sensitivity and dynamic range were evaluated by amplifying a 9-log range of synthetic template concentration in a background of cDNA prepared either from purified RNA or from a TaqMan Cells-to-CT lysate. The data show equivalent target detection over the entire range of input template amounts, indicating that there are no inhibitory or negative effects of the lysate on the sensitivity of the PCR (Figure 2).
Figure 2. TaqMan® Fast Cells-to-CT™ Lysates and Purified RNA Give Comparable Results in Real-Time PCR. A 50 µL RT reaction was performed that contained either 10 µL of TaqMan Cells-to-CT Kit lysate (10,000 NIH/3T3 cells in 50 µL original lysate) or equivalent amounts of purified RNA. A constant volume (20% total PCR volume) of the RT reactions was combined with the indicated amounts of diluted synthetic target for the Hs00430940_m1 assay and analyzed using the assay specific for that target. Results are virtually indistinguishable between reactions performed in a background of purified RNA vs. TaqMan Cells-to-CT Kit lysate.
Compatibility
The TaqMan Fast Cells-to-CT Kit is compatible with multiple cells lines from a variety of tissues (both adherent and suspension). Over 163 TaqMan Gene Expression Assays have been tested, and the CT values obtained using lysates made with the TaqMan Fast Cells-to-CT Kit were essentially equivalent to CT values obtained from use of purified RNA for both high- and low-abundance targets (Figure 3).
Figure 3. High Correlation of Real-Time PCR Results Between TaqMan® Fast Cells-to-CT™ Kit Lysates and Purified RNA. TaqMan Fast Cells-to-CT Kit lysates and purified RNA from equivalent numbers of 5 pooled cell lines were prepared in parallel and evaluated with 163 TaqMan Gene Expression Assays on a 7500 Fast Real-Time PCR System. The CT values obtained from the TaqMan Fast Cells-to-CT Kit lysates were plotted against the CT value for the same assays using purified RNA.
The kit is also compatible with the TaqMan Cells-to-CT Control Kit. The control kit contains a TaqMan Gene Expression Assay for the endogenous gene β-actin, to normalize for sample loading variability, and an artificial XenoRNA™ Control and corresponding TaqMan Gene Expression Assay, to monitor the effects of PCR inhibition (see article, Ideal Control for PCR Inhibition and Cell Input).
The kit is also compatible with the TaqMan Cells-to-CT Control Kit. The control kit contains a TaqMan Gene Expression Assay for the endogenous gene β-actin, to normalize for sample loading variability, and an artificial XenoRNA™ Control and corresponding TaqMan Gene Expression Assay, to monitor the effects of PCR inhibition (see article, Ideal Control for PCR Inhibition and Cell Input).
