452 Housekeeping gene selection and validation for qPCR analysis of psoriasis biopsies

Abstract

Real-time PCR is the “gold standard” of the existing methods for assessing the gene expression. However, despite the accuracy of the methodology as a whole, the results obtained by this method can vary greatly depending on the selected normalization method. Despite the general stability, the expression profiles of the housekeeping genes can vary greatly depending on the type of sample being analyzed. Thus, for each specific task - for example, different types of cells and tissues - it is necessary to select the optimal housekeeping genes that could be used as the reference ones. In this study, based on the data obtained from the RNA-seq of 14 pairs of biopsies taken from lesional and non-lesional skin of psoriasis patients, 14 genes were selected as the candidate reference genes in the analysis of expression profiles. This were BABAM1, GAPDH, ANAPC5, UIMC1, TAX1BP1, POLS3C, OS9, NRD1, XRCC1, USP16, SAP18, NMT1, GGNBP2, CES. Expression profiles of the selected genes were evaluated by the quantitative real-time PCR at an additional panel of 8 pairs of biopsies taken from lesional and non-lesional skin of psoriasis patients. The resulting expression data was analyzed using Bestkeeper, GeNorm, Normfinder services in order to identify the most stable reference genes for psoriatic tissues. Based on the results of the analysis, the genes NMT1, BABAM1, XRCC1 proved to be the most stable, and the most variable ones were TAX1BP1, NRD1 and GAPDH -the gene widely used in the analysis of qPCR data. The data obtained highlights the importance of choosing the right housekeeping genes for each specific cell or tissue type prior to the performance of the expression studies.