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Quantitative real-time reverse-transcription PCR (RT-qPCR) is presently the method of choice for validating gene expression results from high-density microarrays. However, the low throughput of traditional gene-by-gene RT-qPCR makes this process labor intensive and time consuming. To accelerate this laborious task, the SuperArray RT²Profiler PCR Array combines SYBR Green–based real-time RT-qPCR technology with a multi-gene array plate format to simultaneously analyze a panel of genes related to a specific disease or biological pathway.
Each assay on the PCR array plate has been experimentally validated to insure gene-specific amplification. The reliability and reproducibility of the RT²Profiler PCR Array have been demonstrated by DNA sequencing and intra/inter-laboratory reproducibility comparisons. The DNA sequencing demonstrated 100% of the PCR products amplified from the correct target genes. In one laboratory, individual PCR assays produced a standard deviation of 0.24 cycles and a coefficient of variance of 0.92% in technical replicates. The correlation coefficient for Ct values between replicate runs was 0.997 and for fold changes (ΔΔCt) across thermocyclers was 0.976. Comparisons between two different laboratories using different thermocyclers showed correlation coefficients of 0.972 and 0.976 for ΔCt and ΔΔCt, respectively. Each PCR array also includes stringent controls to monitor RNA quality by assessing reverse transcription efficiency and genomic DNA contamination to ensure the reliability of the PCR array data.
A practical application for the PCR array was demonstrated by identifying human pancreatic tumor–associated genes using the Cancer PathwayFinder RT²Profiler PCR Array. Results showed 23 genes exhibiting a statistically significant threefold or greater change in expression between a human pancreatic tumor and normal pancreas, including many genes previously linked to pancreatic cancer. Hence, the RT2 Profiler PCR Array system offers a simple, reliable, and convenient tool for multi-gene profiling and microarray data validation.