Eliminate RNA isolation without compromising the sensitivity or specificity of real-time PCR assays
Overview of Kits and Applications
NEW TaqMan Cells-to-CT Control Kit. The new TaqMan Cells-to-CT Control Kit provides controls for inhibitors of RT-PCR and validation of sample input. This kit includes XenoRNA™ Control, a synthetic RNA transcript with a unique sequence that lacks homology to current, annotated biological sequences, and two TaqMan Gene Expression Assays—for XenoRNA Control and b-actin, a housekeeping gene with moderately abundant expression levels in many cell types.
Applications. The TaqMan Gene Expression Cells-to-CT Kit and the TaqMan Cells-to-CT Control Kit can be used in any application in which real-time RT-PCR is used to analyze mRNA from cultured cells, particularly for large experiments. Applications include real-time PCR analysis of gene expression in cell cultures treated with a library of chemical compounds or with increasing concentrations of a chemical compound, RNAi studies using siRNA to modulate gene expression, and time course experiments.
Figure 1. Simple and Quick Lysis Procedure Using the TaqMan® Gene Expression Cells-to-CT™ Kit. Cultured cells can be lysed directly in 96- and 384-well culture plates or in standard microcentrifuge tubes. In just 7 minutes at room temperature, with no centrifugations or sample transfer steps, cultured cell lysates are ready for RT-PCR using the included RT reagents and TaqMan Gene Expression Master Mix (TaqMan Assays are available separately).
Simple 7-Minute Sample Preparation
The simple, three-step lysis procedure is outlined in Figure 1. Start with 10–105 cultured cells grown in any format, including 96- or 384-well plates, and wash with PBS. Then add the supplied Lysis Solution with or without DNase (included), and incubate for 5 minutes at room temperature to lyse the cells and remove gDNA. Next add Stop Solution and incubate for 2 minutes at room temperature–and you're done! Samples can be processed directly in the culture well, minimizing sample handling and the potential for sample loss or transfer-error, and facilitating high throughput processing. In just 7 minutes, Cells-to-CT lysates are ready for real-time RT-PCR or storage at –20ºC or –80ºC for at least 2 months. XenoRNA™ Control from the Cells-to-CT Control Kit can be added to the Stop Solution just before addition to samples to provide a positive control target for experiments.
Reliable Quantitation of Both Limited and Abundant Targets
The TaqMan Gene Expression Cells-to-CT Kit includes reverse transcription (RT) reagents optimized for highly efficient cDNA synthesis from Cells-to-CT lysates, and formulated for convenience. Simply incubate the lysate (up to 45% of the final reaction volume) with the RT enzyme mix and buffer. The cDNA is then ready for real-time PCR using TaqMan Gene Expression Master Mix (supplied with the kit) and the TaqMan Gene Expression Assay for the target of interest (available separately).
For real-time PCR, the new Applied Biosystems TaqMan Gene Expression Master Mix delivers extremely sensitive target detection across a broad range of template quantities and high specificity for discrimination among gene family members. Highly purified AmpliTaq Gold®, UP DNA Polymerase powers the master mix, which is formulated for reliability and consistency even in challenging applications, such as precise quantitation of low and similar (<2-fold) target quantities, and amplification of targets with AT- or GC-rich regions.
Figure 2. Correlation of Real-Time PCR Results Between Cells-to-CT™ Lysates and Purified RNA. Cells-to-CT lysates and purified RNA from HeLa cells were prepared in parallel and evaluated with 128 TaqMan® Gene Expression Assays on an Applied Biosystems 7900HT Real-Time PCR System. (A) The CT value obtained from the Cells-to-CT lysates is plotted against the CT value for the same assay using purified RNA. R2 = 0.9107 for these assays. Detailed amplification plots for selected assays from Cells-to-CT lysates (B) and purified RNA (C) are shown for 5 TaqMan Assays.
Compatibility with a Wide Range of Cell Types
Figure 3. Cell Types Compatible with the TaqMan® Gene Expression Cells-to-CT™ Kit.
Use the TaqMan Cells-to-CT Control Kit for Routine Positive Controls and for Pilot Experiments with New Cell Types
The TaqMan Cells-to-CT Control Kit can also be used to evaluate whether untested cell lines are compatible with the TaqMan Gene Expression Cells-to-CT Kit and to identify the maximum number of cells that can be used per Cells-to-CT lysis reaction. For these investigations, we recommend serially diluting cells and preparing a lysate that contains the XenoRNA Control. Then, use each lysate in parallel RT-PCRs with the TaqMan assays for both the XenoRNA Control and beta-actin. Depending on the experimental goals, it may also be useful to include a third RT-PCR targeting the gene of interest to evaluate the lower limits of cell input for target detection. An example of this type of experiment is shown in Figure 4.
Figure 4. Pilot Experiment Identifies Maximum Number of Cells per Lysis Reaction. By adding XenoRNA™ Control to the Stop Solution used to prepare TaqMan® Gene Expression Cells-to-CT™ lysates, the XenoRNA TaqMan Assay can be used to evaluate increasing amounts of cells per lysis reaction for inhibitors of RT-PCR. In parallel, the cDNA from these same lysates can be amplified using the beta–actin assay to verify that sample lysis is adequate and that cellular RNA is available for RT-PCR.
The TaqMan Gene Expression Cells-to-CT Kit includes reagents for 100 lysis reactions with gDNA removal, 100 RT reactions, 100 minus-RT control reactions, and either 200 PCRs (50 µL reaction volume) or 500 PCRs (20 µL reaction volumes). As listed in the Ordering Information, a 400 lysis reaction kit is also available.
Laura Chapman, Annalee Nguyen, Richard Fekete • Applied Biosystems, Austin, TX
Junko Stevens, Shoulian Dong, Chunmei Liu • Applied Biosystems, Foster City, CA