|
TaqMan® Gene Expression Cells-to-CT™ Kit
TaqMan® Arrays
Streamlined Expression Analysis of Multiple Genes Using Cell Lysates with TaqMan® Arrays
The combination of TaqMan® Gene Expression Cells-to-CT™ Kit for sample preparation with TaqMan Arrays (micro fluidic cards pre-loaded with up to 384 real-time RT-PCR assays) provides researchers with a fast and accurate workflow for gene expression analysis from cultured mammalian cells. As shown here, when gene expression is assessed with TaqMan Arrays, cell lysates from the TaqMan Gene Expression Cells-to-CT Kit performed equivalently to purified RNA for most samples and often showed improved sensitivity for samples with very low cell numbers.
Gene Expression Analysis Without RNA Isolation
The TaqMan Gene Expression Cells-to-CT Kit makes it possible to perform expression analysis directly from cultured cells without RNA purification. This innovative sample preparation procedure features a unique method for lysing cultured cells while removing genomic DNA and preserving RNA integrity. Additionally, 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. These components allow users to test their samples directly with TaqMan Gene Expression Assays and TaqMan Arrays (see sidebar, TaqMan Arrays for Easy and Robust Gene Expression Analysis).
In this study, samples were prepared with the TaqMan Gene Expression Cells-to-CT Kit and an RNA purification kit. After reverse transcription, the samples were loaded on TaqMan Arrays (TaqMan Endogenous Control Array), and the results from cell lysates versus purified RNA were compared.
Sample Preparation
Sample Set 1: Cell Lysates Using the TaqMan Gene Expression Cells-to-CT Kit. HeLa cells (10–100,000 cells) were washed with PBS and lysed for 5 minutes at room temperature; DNase treatment was performed simultaneously (Figure 1). Lysis was terminated at room temperature by a 2-minute incubation with Stop Solution.
|
|
|
| Figure 1. A Simplified Workflow for Analyzing Gene Expression Using TaqMan® Gene Expression Cells-to-CT™ Kit Lysates and TaqMan® Arrays. |
Lysates may be frozen for up to 5 months or can be added directly to the RT reaction. 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 multi-step RNA isolation protocols, no heating, washing, or centrifugation steps are required. The kit greatly simplifies a 30–60 minute process to 7 minutes.
Sample Set 2: RNA Purification Using Column-Based Protocol. Total RNA was purified from HeLa cells (10–100,000 cells) using a commercially available, column-based kit.
Reverse Transcription (RT). After sample preparation, lysates or purified RNA were combined with the 2X RT Buffer and 20X RT Enzyme Mix for RT. In these experiments (Figures 2–3), sample comprised 20% of the RT reaction volume; however, up to 45% of the RT reaction can be composed of lysate, maximizing sensitivity and sample carry-through.
|
|
|
| Figure 2. Performance of TaqMan® Gene Expression Cells-to-CT™ Kit Lysates is Equivalent to or Better Than Purified RNA. Lysates or purified RNA samples generated from 10–100,000 HeLa cells were reverse transcribed, combined with TaqMan Gene Expression Master Mix, and loaded onto TaqMan Human Endogenous Control Arrays. Six of the 16 genes tested using the Endogenous Control Array are shown as representative results. |
|
| Figure 3. The Slope and Squared Linear Correlation Coefficients for TaqMan® Gene Expression Cells-to-CT™ Kit Lysates Are Near Ideal. Lysate or purified RNA generated from 10–100,000 HeLa cells was reverse transcribed, combined with TaqMan Gene Expression Master Mix, and loaded onto Human Endogenous Control Panel TaqMan Arrays. Slopes of CT values versus log(cell number) (A) and squared linear correlation coefficients (R2) (B) were calculated for each assay over the range of cell numbers. |
TaqMan Arrays for Gene Expression Analysis
Master Mix. Using the TaqMan Gene Expression Master Mix included in the TaqMan Gene Expression Cells-to-CT Kit, cDNA can comprise up to 45% of the real-time PCR volume. This master mix amplifies the target precisely and accurately, enabling the detection of small quantities of template, such as transcripts expressed at low levels.
Real-Time PCR. After RT of cell lysates and purified RNA, the resulting cDNA samples were combined with TaqMan Gene Expression Master Mix and loaded onto TaqMan Human Endogenous Control Arrays. RT products comprised 20% of the PCR volume for each sample. The Endogenous Control Arrays contain primers/probes for 16 commonly used housekeeping genes and genes that exhibit minimal differential expression across a large number of tissues. The arrays were run using universal thermal cycling conditions on a 7900HT Fast Real-Time PCR System.
Results and Conclusions. The performance of lysates generated with the TaqMan Gene Expression Cells-to-CT Kit was equivalent to or surpassed that of purified RNA. For many samples, especially those at low cell numbers, the sensitivity of assays using lysates was superior to that of purified RNA, likely due to sample loss during RNA isolation methods that use spin columns (Figure 2).
In addition to greater sensitivity at low cell numbers, lysis efficiency is maintained at high cell numbers as shown by the slopes of the data, which have a value close to the ideal of –3.3 (Figure 3A). Poor efficiency (slope >3.5) seen with the traditional RNA isolation samples is likely due to inefficient cell lysis and sample recovery. A squared linear correlation coefficient close to 1.0 is observed for all assays (Figure 3B).
The TaqMan Gene Expression Cells-to-CT Kit and TaqMan Arrays provide a complete workflow for optimal simplicity and speed with robust results customers expect from Applied Biosystems products. A complete list of available TaqMan Gene Signature Arrays is available on the Applied Biosystems website.
Scientific Contributors
Annalee Nguyen, Laura Chapman, Richard Fekete • Applied Biosystems, Austin, TX
David Keys • Applied Biosystems, Foster City, CA
|
