Validating affymetrix chip results with real time pcr

We selected 47 genes represented on both arrays, including both known regulated and unregulated transcripts, and established reference relative expression measurements for these genes in the test RNA samples using quantitative reverse transcriptase real-time PCR (QRTPCR) assays.

The validity of the reproducible (average coefficient of variation = 11.8%) QRTPCR measurements were established through application of a new mathematical model.

Accuracy of the fold-change measurements obtained with each platform was assessed by determining measurement bias.

validating affymetrix chip results with real time pcr-76

All alternative splicing events selected for validation were confirmed by real-time PCR.We show excellent inter-platform concordance between Affymetrix microarray and Veri Quest real-time PCR data.We compared the accuracy of microarray measurements obtained with oligonucleotide arrays (Gene Chip, Affymetrix) with a laboratory-developed c DNA array by assaying test RNA samples from an experiment using a paradigm known to regulate many genes measured on both arrays.This study thus represents a thorough validation of the GWA approach.It has also demonstrated that careful use of a shared control group represents a safe and effective approach to GWA analyses of multiple disease phenotypes; has generated a genome-wide genotype database for future studies of common diseases in the British population; and shown that, provided individuals with non-European ancestry are excluded, the extent of population stratification in the British population is generally modest.

Validating affymetrix chip results with real time pcr