The Ambion® TaqMan PreAmp Cells-to-CT Kit makes it possible to perform gene expression analysis directly from limited or small numbers of cultured cells without RNA purification. The protocol includes an intermediate amplification step (pre-amplification) between reverse transcription and real-time PCR in which cDNA is enriched for up to 100 gene targets of the user’s choice without amplification bias.
• Complete, validated solution—Optimized workflow includes cell lysis reagents, DNase, RT reagents, PreAmp reagents, and TaqMan® Gene Expression Master Mix • Fast, simple and convenient—Prepare samples in 7 minutes; eliminate tedious RNA isolation • Extend limited samples—Increase real-time PCR potential by up to 64-fold; study up to 100 genes at once • Accurate results—Uniform amplification without introducing bias • Robust performance—Linear detection from 10 to 100,000 cells; results equivalent to purified RNA
Complete, Validated Solution
Featuring a unique method for lysing cultured cells while removing genomic DNA and preserving the RNA integrity, the TaqMan® PreAmp Cells-to-CT™ Kit contains reverse transcription (RT) reagents for cDNA synthesis, TaqMan® PreAmp Master Mix for pre-amplification of up to 100 gene targets, and TaqMan® Gene Expression Master Mix for real-time PCR analysis.
Simple, Convenient 7-Minute Sample Preparation
The TaqMan® PreAmp Cells-to-CT™ Kit incorporates a simple 7-minute sample preparation procedure (Figure 1) for use with 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 concurrently. 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 plates (96 or 384 wells), sample handling and the potential for sample loss or transfer error are minimized, facilitating extension of limited samples. No heating, washing, or centrifugation is required; the kit greatly simplifies a traditionally time-consuming, labor intensive process and reduces it to 7 minutes.
Extension of Limited Samples—Increase Real-Time PCR Potential by up to 64-fold
The integration of the Cells-to-CT™ technology with pre-amplification capability streamlines a multi-step workflow while significantly extending (greater than 60-fold) the number of PCR reactions possible from precious samples, and increases the sensitivity of mRNA detection from small amounts of sample input.
The standard real-time PCR reaction for gene expression analysis starts with the reverse transcription of total RNA to cDNA using random primers and oligo dT, followed by real-time PCR using gene-specific primers and probes (Figure 2). With the TaqMan® PreAmp Cells-to-CT™ Kit, an intermediate amplification step (pre-amplification) between reverse transcription and real-time PCR is performed in which cDNA is enriched for up to 100 gene targets of the user’s choice without amplification bias. The resulting pre-amplified reaction is diluted and serves as the starting material for the subsequent individual TaqMan® Gene Expression Assays using the TaqMan® Gene Expression Master Mix, included in the kit. The TaqMan® PreAmp process effectively extends samples up to 64-fold, enabling analyses previously not possible from limited sample material.
Accurate Results—Equal Amplification without Introducing Bias
The ability to conduct reliable and uniform pre-amplification is critical for gene expression analysis from limited samples. While other methods are available to pre-amplify RNA or cDNA, they use non-specific priming to amplify the starting material, resulting in biased and inaccurate amplification of some transcripts over others. The TaqMan® PreAmp Master Mix, part of the TaqMan® PreAmp Cells-to-CT™ Kit, uses gene specific primers to pre-amplify cDNA resulting in unbiased amplification. The TaqMan® PreAmp Cells-to-CT™ Kit demonstrates unbiased pre-amplification equivalent to purified RNA when compared across 100 different TaqMan Gene Expression Assays. As shown in Figure 3, the pre-amplification reaction does not introduce bias from Cells-to-CT™ lysates nor purified samples; ≥ 92% of the assays were within +⁄- 1.5 ΔΔ CT for pre-amplified vs. un-amplified cDNA from either purified RNA (A) or from Cells-to-CT™ Kit lysates (B).
Robust Performance—Results Equivalent to Purified RNA Performance of the TaqMan® PreAmp Cells-to-CT™ Kit was evaluated with 100 different TaqMan® Assays utilizing Cells-to-CT™ lysates or purified RNA, either with or without pre-amplification. As shown in Figure 4, the CT values obtained with TaqMan® PreAmp Cells-to-CT™ Kit lysates were equivalent to, and frequently better than CT values obtained using purified RNA, either prior to (A) or after pre-amplification (B). The high level of performance from TaqMan® PreAmp Cells-to-CT™ Kit lysates can be attributed to the preservation of RNA in the starting samples without loss from heating, transfer, or adsorption to matrices.
Linear Detection from 10 to 100,000 Cells
Each step of a workflow for profiling RNA must be robust to work from a few, or many cells. The TaqMan® PreAmp Cells-to-CT™ Kit has been optimized to efficiently generate linearity across 5 logs of cellular input, from 10–100,000 cells per lysis reaction (Figure 5). Furthermore, to maximize sensitivity, the TaqMan® PreAmp Cells-to-CT™ Kit allows up to 45% of the RT reaction volume to be comprised of sample lysate. The TaqMan® PreAmp Cells-to-C
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