Validating internal controls for quantitative plant gene expression
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.1 2, Fax 1 2, E-Mail [email protected]: Endogenous internal controls (‘reference’ or ‘housekeeping’ genes) are widely used in real-time PCR (RT-PCR) analyses.Renal expression levels of 16 commonly used endogenous controls for RT-PCR analyses were determined in 2 groups of age-matched cpk mice generated in a (C57BL/6J-cpk × CAST/Ei)F1 intercross : 7 mice with the mildest cystic kidney phenotype and 7 mice with the most severe cystic kidney phenotype.The expression levels were determined in triplicates by Taq Man®-based RT-PCR assays (online suppl. These 16 genes were also included in our previously conducted microarray experiments with Affymetrix Gene Chip® arrays in these renal tissues .The overall variance, a basis of a simple, yet highly effective, tool for the identification of ‘reference genes’ , was calculated for the normalized C values of each of the 3 technical replicates.The overall variances were compared across the 16 ‘reference genes’ to identify optimal RT-PCR controls with the most stable gene expression (smallest overall variance) across all kidney samples (fig. For example, 18S r RNA and Hmbs showed the largest variation for all 3 replicates, indicating that these genes represented suboptimal endogenous controls in this experimental setup.Their use relies on the premise of consistently stable expression across studied experimental conditions.
Expression levels of these 16 genes, determined by Taq Man® RT-PCR analyses and Affymetrix Gene Chip® arrays, were normalized and tested for overall variance and equivalence of the means.Comparisons of data generated by these 2 distinct gene expression platforms allowed an independent validation of these 2 approaches.First, normalization of the Taq Man®-based RT-PCR data was performed for all samples by subtracting the mean of the C values, this approach successfully reduced variances in the expression of all studied genes, although to various extents (fig. Then, we ranked the appropriateness of the 16 ‘reference genes’ as endogenous internal RT-PCR controls based on their overall variance, with smaller variance indicating higher gene expression stability.The assumption for this normalization is that the total signals from all studied ‘reference genes’ are the same across all samples.The preprocessing of the microarray data analysis was performed as previously described .