Selection of reference genes for mRNA quantification in Trypanosoma brucei.

Internal normalization is an established procedure that is necessary for accurate and reliable quantification of differentially regulated mRNAs. The profound changes of gene expression in parasitic life cycles pose a particular challenge on selection of appropriate reference genes for normalization, most importantly when using quantitative real time PCR (qPCR). Here we use the ranking algorithm implemented in the geNorm application to identify suitable Trypanosoma brucei reference genes for comparisons between the bloodstream and procyclic developmental stages and for analysis of mRNA induction by environmental conditions. For these conditions, the TERT gene is a good choice for valid normalization of qPCR and is clearly superior to some other reference genes reported in the literature. For comparison of other conditions, the ranking algorithm is recommended to verify a reliable and valid normalization that is instrumental to quantitative analysis of gene expression.