Endogenous control genes in complex vascular tissue samples

In complex tissue, microarray data and real-time PCR data show the best correlation when endogenous control genes are omitted and the normalization is done relative to total RNA mass, as measured before reverse transcription.We have found that for real-time PCR in heterogeneous tissue samples, it may be a better choice to normalize real-time PCR Ct values to the carefully measured mass of total RNA than to use endogenous control genes. We base this conclusion on the fact that total RNA mass normalization of real-time PCR data shows better correlation to microarray data. Because microarray data use a different normalization approach based on a larger part of the transcriptome, we conclude that omitting endogenous control genes will give measurements more in accordance with actual concentrations.Real-time PCR is a sensitive method for expression analysis widely used for both cell culture and complex tissues. Relative quantification of mRNA levels using real-time PCR data is commonly done using the 2^(-ΔΔCt) method [1]. A central idea of this method is the use of an endogenous control for normalization, a so-called housekeeping gene. The aim of this normalization is to correct for different amounts of starting material of RNA or differences in the cDNA synthesis efficiency. Commonly used selection criteria for housekeeping genes are genes with the least amount of variance across all samples and genes that show no trends of change in relation to sample parameters of interest. However, because of lack of methods to determine low variance - other than real-time PCR itself - the selection of endogenous controls often comes precariously close to circular reasoning. Vandesompele and coworkers have suggested methods to circumvent this, through the iterative calculation of pairwise correlations with other potential endogenous control genes and removal of the most deviating candidates [2].To investigate the merit of these endogenous control selection methods, we analyzed gene e