Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD

Abstract

Background: Selection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: b-actin, glyceraldehyde-3-phosphate dehydrogenase, b2-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD. Results: The variability in expression of the four endogenous controls was assessed by TaqMan Real-time RT-PCR in peripheral blood mononuclear cells from: war veterans with current PTSD, those with lifetime PTSD, trauma controls and healthy subjects. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified b-actin and glyceraldehyde- 3-phosphate dehydrogenase as a pair of genes with the lowest stability value. Conclusions: The combination of b-actin and glyceraldehyde-3-phosphate dehydrogenase appeared to be the most suitable reference for studying alterations in gene expression in peripheral blood mononuclear cells related to vulnerability and resilience to PTSD, as well as to trauma-provoked developing of this disorder and recovery from it. Using glyceraldehyde-3-phosphate dehydrogenase, b-actin and b2-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended.