TaqMan™ Fast Cells-to-C_T™ Kit (Invitrogen™)

The TaqMan™ Fast Cells-to-C_T™ Kit makes it possible to use Fast-cycling, real-time PCR chemistry and instrumentation to perform gene expression analysis directly from cultured cells without RNA purification.

The TaqMan™ Fast Cells-to-C_T Kit is:

• Complete—optimized workflow includes cell lysis reagents with gDNA removal, RT enzyme mix, RT buffer, and TaqMan™ Fast Universal PCR Master Mix
• Fast—prepare samples at room temperature in 7 minutes, including DNase treatment; TaqMan™ Fast Universal PCR Master Mix delivers real-time PCR results in ∼35 minutes
• Convenient—lyse samples in a tube or directly in 96- or 384-well culture plates
• Robust—perform gene expression analysis on 10 to 100,000 cells per sample, with results equivalent to those from purified RNA
• Efficient—contains sufficient reagents to generate 500 real-time PCR results from 100 starting samples

Complete, validated solution
TaqMan™ Cells-to-C_T™ technology features a unique method for lysing cultured cells while removing genomic DNA (gDNA) and preserving RNA integrity. Additionally, the kit contains reverse transcription (RT) reagents and TaqMan™ Fast Universal PCR Master Mix for Fast real-time PCR analysis, and has been extensively tested and validated with gold-standard TaqMan™ Gene Expression assays, sold separately. All kit components have been optimized to work together for consistent and reliable performance, removing the guesswork involved in assembling separate kits for sample preparation, reverse transcription, and Fast real-time PCR.

Fast, convenient workflow
The TaqMan™ Fast Cells-to-C_T™ Kit incorporates a simple 7-minute sample preparation procedure (see figure) for use with 10 to 100,000 cultured cells directly in 96- or 384-well culture plates or in tubes. Cells are washed in PBS and lysed for 5 minutes at room temperature; an optional DNase treatment can be performed concurrently. Lysis is terminated at room temperature by a 2-minute incubation with Stop Solution. The kit greatly simplifies a traditionally time-consuming, labor intensive process and reduces it to 7 minutes.

The lysates are now ready for reverse transcription with the optimized RT system included in the kit, or they can be stored at -20°C for up to 5 months, for future analysis. The supplied TaqMan™ Fast Universal PCR Master Mix delivers sensitive and reproducible results approximately 3 times faster than standard PCR reagents and instruments. The combination of fast and simple sample preparation, and Fast-cycling real-time PCR results (∼35 minutes) enables more rapid quantitation of gene targets than previously available. The TaqMan™ Fast Cells-to-C_T™ Kit workflow reduces the time from sample to result by 40% or more, compared to traditional RNA purification and standard real-time PCR.

Robust, sensitive, and reliable results
Accurate gene expression analysis over a wide range of sample input for genes with various expression levels has traditionally been a challenge for researchers. Therefore, a robust solution is important for profiling RNA from a few or many cells. Shown is the demonstration of the excellent signal linearity (R2 = 0.997) observed using lysates from TaqMan™ Fast Cells-to-C_T™ Kit across 5 logs of cellular input (10–10⁵ cells per lysis reaction) (see figure). Thus, a single kit can be used for analysis of gene expression from limited or dense samples, with no compromise in data integrity caused by PCR inhibitors.

PCR performance of cell lysates using the TaqMan™ Fast Cells-to-C_T™ Kit was equivalent to that from purified RNA, using the supplied TaqMan™ Fast Universal PCR Master Mix (see figure). Both TaqMan™ Cells-to-C_T™ Kit lysates and purified RNA showed linear detection over a 5 log range of cell input equivalents. The enhanced sensitivity at the 10-cell input with TaqMan™ Fast Cells-to-C_T™ Kit lysates compared to purified RNA, using the same RT and PCR reagents, results from more efficient retention of target molecules during preparation, i.e., no loss of RNA typically associated with sample transfer or heating.

The TaqMan™ Fast Cells-to-C_T™ Kit also demonstrated superior performance compared to purified RNA which was reverse transcribed and PCR amplified with Competitor Q’s RT and Fast Master Mix. The observed dynamic range using Competitor Q’s reagents with Fast cycling was 10² to 10⁵ cell equivalents, or 1 log less than TaqMan™ Cells-to-C_T™ Kit lysates or purified RNA amplified with TaqMan™ Fast Universal PCR Master Mix (see figure). The TaqMan™ Fast Universal PCR Master Mix is formulated to deliver sensitive and reproducible results faster than is possible with standard reagents and instruments, and is robust across a wide range of target genes and concentrations.