Real-time PCR is an extremely versatile tool to analyse gene expression: the selection of stably expressed reference genes is a crucial step which inevitably affects data reliability.
Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample.
Stepwise exclusion of the gene with the highest M value results in a combination of two constitutively expressed reference genes that have the most stable expression in the tested samples.
To get more reliable quantification results, we performed an experiment in advance to screen reference genes for qPCR (see Methods for details), and the relative expression levels of unigenes were normalized to 3 stable expressed reference genes.
Therefore, it is of paramount importance to find stably expressed reference genes, as the reliability of the normalised expression data is only as good as the reliability of the reference gene(s).
Reliable and accurate expression data can only be obtained by normalization with stably expressed reference genes.
Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as an endogenously expressed reference gene for the purpose of quantifying relative gene expression.
The stepwise exclusion of the reference gene with the least stable expression showed that ACTB and TOP2B were the most stably expressed reference genes in the analyzed samples.
Various strategies have been explored in an attempt to normalize these variations, and it is generally accepted that gene-expression levels should be normalized by carefully selected and stably expressed reference genes [ 2- 4].
TBP was chosen for the reference gene, because there are no known retropseudogenes for it and its expression is lower than that of many commonly used abundantly expressed reference genes (Bieche et al, 1999).
The most consistently expressed reference genes in each tissue were identified and used to normalize gene expression data.
