Validating internal controls for quantitative plant gene expression

25-Oct-2016 13:16 by 7 Comments

Validating internal controls for quantitative plant gene expression

Methods: To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used ‘reference genes’ in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model.

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.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 [15].severe and mild kidney disease) from the within-group variance.In other words, a small overall variance does not necessarily imply small fold changes between the 2 groups.In contrast to the previously utilized 2-fold change threshold for the equivalence test [6], we used a more stringent 1.5-fold change threshold since a 2-fold change is often assumed to be differentially expressed and not appropriate to consider as a ‘reference gene’.

The overall variance analysis was conducted on the same data in the same fashion as the equivalence test except that the overall variances of all samples were computed for each gene.However, the ranking was not affected by the normal assumption.For the RT-PCR data, 1 technical replicate was arbitrarily chosen from each sample for the equivalence test.For the microarray data, each probe set was analyzed separately.The median p value from each gene was used for overall gene ranking.In contrast, Ppia, Gapdh and Pgk1 showed the smallest variation and would serve as the more appropriate endogenous internal RT-PCR controls.