principles of thermoluminescence dating - Validating microarray data with real time rt pcr
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.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.
The performance of both array platforms in identifying regulated and non-regulated genes was identical.
Both platforms consistently underestimate the relative changes in m RNA expression between experimental and control samples.
The bias observed with c DNA arrays was predictable for fold-changes We describe detailed protocols and results with an integrated platform for studying relative transcript expression, including microarray design and fabrication, analysis and calibration algorithms, and high throughput quantitative real-time PCR.
This focused array facilitated both repeated measurement and replicate experiments.