Validating microarray data with real time rt pcr

Xiangqin Cui, Ph D and Michal Mrug, MDDivision of Nephrology, University of Alabama at Birmingham1900 University Blvd, Tinsley Harrison Tower 611JBirmingham, AL 35294 (USA)Tel.

Endogenous internal controls, often referred to as ‘housekeeping’ or ‘reference’ genes, offer multiple practical advantages and are therefore widely used in research and clinical applications.Their use, however, relies on the premise that these genes are expressed at consistently stable levels across all experimental conditions under investigation.Unfortunately, none of the endogenous controls was found to be constantly expressed across different tissues, developmental stages, or pathological and study conditions [1, 2].The suitability of individual ‘reference genes’ as internal RT-PCR controls for a specific experimental design can be tested with various approaches, including methods of overall variance [3], ANOVA models [4], Bayesian models [5] and equivalence testing [6, 7].

More recently, genome-wide expression technologies have been recognized as an effective tool for the identification and evaluation of endogenous RT-PCR controls [8, 9].

Even more accurate RT-PCR controls could be obtained by accounting for the expression of multiple genes, e.g.