Using Excess Labeled aRNA for Microarray Validation
|Our scientists demonstrate that biotin- or amino allyl-labeled aRNA (amplified RNA) synthesized with MessageAmp™ II aRNA Amplification Kits can be used in TaqMan® Gene Expression Assays. This means that the excess amplified RNA not needed for microarray profiling experiments can be used to pre-screen microarray samples or to validate microarray results.|
- MessageAmp II aRNA Amplification Kit, for synthesis of unlabeled aRNA
- MessageAmp II-Biotin Enhanced Kit, for synthesis of biotinylated aRNA
- Amino Allyl MessageAmp II aRNA Amplification Kit, for synthesis of amino allyl-labeled aRNA
The resulting aRNAs were unmodified RNA, RNA labeled with biotin-11-UTP, or RNA modified with amino allyl UTP. aRNA samples were purified according to protocol. TaqMan Gene Expression Assays for seven housekeeping genes (GAPDH, B2M, GUSB, HPRT1, PGK1, PPIA and RPLP0) were used for the RT-PCR analysis. Each aRNA sample (5 ng) was assayed in quadruplicate with TaqMan Assay using TaqMan One-Step RT-PCR Master Mix from Applied Biosystems.
The TaqMan Assay reactions were performed in a 7900HT Real Time PCR System (Applied Biosystems), programmed as follows: 48°C for 30 minutes, 95°C for 5 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds. The average Ct values for unlabeled, biotin-labeled, and amino allyl-labeled aRNAs were determined.
Figure 1. Effect of aRNA Labeling Method on TaqMan® One-Step qRT-PCR Reactions. Duplicate MessageAmp™ II reactions for each of the RNA amplification labeling methods (no labeling, biotin labeling, amino allyl labeling) were performed using 1 µg FirstChoice® Total RNA from HeLa cells. Resulting aRNA (5 ng) was assayed in quadruplicate in one-step qRT-PCR with TaqMan Gene Expression Assays for seven housekeeping genes. Quadruplicates were averaged, then the average of the duplicate MessageAmp II reactions was plotted (mean ±1 SD) for the three labeling methods. TaqMan Gene Expression Assay ID numbers are shown below each gene symbol (X-axis).