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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 Kits routinely yield far more labeled aRNA (amplified RNA, also known as complementary RNA or cRNA) than is needed for microarray profiling. This excess aRNA would prove highly useful for validation of microarray results with TaqMan Gene Expression Assays or as a pre-hybridization QC step prior to committing samples to costly microarray analysis. However, it was not known whether the biotin or amino allyl-label on aRNA would affect TaqMan Assay performance. Here we compare results from TaqMan Assay reactions using biotin- and amino allyl-labeled aRNA in parallel with unlabeled aRNA and demonstrate that researchers can use labeled aRNA from MessageAmp II reactions directly with TaqMan Gene Expression Assays for validation of microarray results.
Experimental Design
FirstChoice Total RNA (1 µg) from HeLa cells was used in each duplicate RNA amplification reaction performed with three MessageAmp II RNA Amplification Kits:
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.
- 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.
Results
We found that the label on aRNA did not inhibit the TaqMan Assays as shown in Figure 1. Similar Ct values were obtained for the three different aRNA types across all seven TaqMan assays. There appeared to be a slight inhibitory effect of the amino allyl label on amplification of aRNA for some genes, although one gene, HPRT1, showed the opposite effect.
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).
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).
Conclusions
Biotin- or amino allyl-labeled aRNA generated from a MessageAmp II reaction is suitable for use directly in TaqMan Assays. Some researchers may find this useful for interrogating a specific gene of interest prior to committing samples to microarray analysis. Others may find that the aRNA synthesized from small or hard-to-obtain samples can be used for both microarray profiling and validation experiments.