Go from Cells in Culture to Real-Time PCR Data Even Faster
Save Time and Effort
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
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.
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).