The Ambion® TaqMan® Gene Expression Cells-to-CT™ Kit makes it possible to perform expression analysis directly from cultured cells without RNA purification. This kit saves time and offers a simple workflow that is suitable for a few samples or can be easily incorporated into automated, high throughput applications.
• Complete solution—Optimized workflow includes cell lysis reagents with gDNA removal, RT enzyme mix, buffer, and new TaqMan® Gene Expression Master Mix
• Fast—7-minute sample prep, including DNase treatment, at room temperature
• Easy—Lyse samples in a tube or directly in culture plates
• Robust—Perform gene expression analysis on 10–100,000 cells per sample; results equivalent to those from purified RNA
• Efficient—Contains sufficient reagents to generate 500 real-time PCR results from 100 starting samples
Featuring a unique method for lysing cultured cells while removing genomic DNA and preserving RNA integrity, the TaqMan® Gene Expression Cells-to-CT™ Kit contains reverse transcription (RT) reagents for cDNA synthesis and TaqMan® Gene Expression Master Mix for real-time PCR analysis. TaqMan® primer⁄probe sets are sold separately.
Simple 7-Minute Sample Preparation: Part of a Complete Workflow
Whether you are using plates or tubes, the TaqMan® Gene Expression Cells-to-CT™ Kit, which uses the simple 7-minute sample preparation procedure outlined in Figure 1, is designed for 10–100,000 cultured cells⁄sample. Cells are washed in PBS and lysed in solution for 5 minutes at room temperature; DNase treatment can be performed simultaneously. Lysis is terminated at room temperature by a 2-minute incubation with Stop Solution. The lysates are now ready for reverse transcription or storage at –20°C. Because samples can be processed directly in culture wells (96 or 384 wells), sample handling and the potential for sample loss or transfer error are minimized, facilitating rapid, high throughput processing. Unlike old-fashioned multi-step RNA isolation protocols, no heating, washing, or centrifugation steps are required. The kit greatly simplifies a laborious 30–60 minute process and reduces it to 7 minutes.
The TaqMan® Gene Expression Cells-to-CT™ Kit workflow enables unsurpassed gene expression evaluation with any of the >700,000 TaqMan® Gene Expression Assays. This new kit has been extensively tested for specificity with a broad selection of TaqMan® Gene Expression Assays and shows performance equivalent to that obtained with purified RNA (Figure 2).
Achieve Unsurpassed Performance and Sensitivity
Unlike some competitor kits that limit the amount of lysate in the RT reaction to 5%, the TaqMan® Gene Expression Cells-to-CT™ Kit can accommodate 45% of the total RT reaction volume as cell lysate. Additionally, cDNA can comprise up to 45% of the real-time PCR reaction volume. The large lysate volume in the optimized RT reaction, along with the large cDNA volume in the subsequent real-time PCR using the TaqMan® Gene Expression Master Mix, lead to maximum sensitivity. The master mix amplifies the target precisely and accurately, enabling the detection of small quantities of target, such as transcripts expressed at low levels.
The ability to detect limited target quantities was tested by mixing a constant number of human cells with various numbers of mouse cells. The cell mixtures were prepared using the TaqMan® Gene Expression Cells-to-CT™ and assayed for a mouse-specific and human-specific gene. Comparative data were generated in the same manner with RNA purified by traditional methodology. The data show that RNA from as few as 10 mouse cells was detectable in a background of RNA from 10,000 human cells (Figure 3). The ability of the TaqMan® Gene Expression Cells-to-CT™ Kit to detect relative low abundance transcripts was equivalent to that of purified RNA; at low levels, it was superior.
Additionally, the performance of the TaqMan® Gene Expression Cells-to-CT™ Kit was compared to competitor lysate kits and to purified RNA. Inputs of 100–100,000 cells⁄lysis reaction were examined. The sensitivity of the TaqMan® Cells-to-CT™ Kit protocol was equivalent to that obtained with purified RNA, and it surpassed competitor lysates (Figure 4). Furthermore, the lack of inhibition at high cellular inputs and the low variation among technical replicates demonstrate the reliability of this approach for gene expression studies using cultured cells.
Simple, Reliable siRNA Experiments
The growing interest in RNAi screening utilizing siRNA-mediated gene silencing is driving the need for innovative tools for this application. The TaqMan® Gene Expression Cells-to-CT™ Kit simplifies detection of siRNA-induced mRNA knockdown via qRT-PCR by eliminating the need for RNA purification without sacrificing the sensitivity and robustness required for reliable results.
In the experiment depicted in Figure 5, HeLa cells were transfected with 20 different individual siRNAs or Silencer® GAPDH Positive Control siRNA. After incubation, samples were prepared for qRT-PCR with either the TaqMan® Gene Expression Cells-to-CT™ Kit or by a traditional magnetic bead based RNA purification method, and gene silencing was subsequently measured using the corresponding TaqMan® Gene Expression Assay. Samples prepared with the TaqMan® Gene Expression Cells-to-CT™ Kit showed results equivalent to purified RNA across all targets, demonstrating the excellent performance of the kit in facilitating analysis of siRNA-mediated transcript knockdown.
Proven Performance, Proven Together
All components of the TaqMan® Gene Expression Cells-to-CT™ Kit have been optimized for consistent and reliable performance. This removes the guesswork involved in assembling separate sample preparation, RT, and real-time PCR kits. And the TaqMan® Gene Expression Cells-to-CT™ Kit has been validated with TaqMan Gene Expression Assays for added quality assurance.
The TaqMan® Gene Expression Cells-to-CT™ Kit provides sufficient reagents for 500 real-time PCRs from 100 starting samples (100 rxn kit).
TaqMan® Cells-to-CT™ Control Kit
Designed for use with the TaqMan® Gene Expression Cells-to-CT™ Kit, this kit contains an endogenous control (ACTB) to normalize for sample loading variability and an artifical XenoRNA™ control and corresponding TaqMan Gene Expression Assay to monitor the effects of PCR inhibition.