Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD.
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: beta-actin, glyceraldehyde-3-phosphate dehydrogenase, beta2-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 beta-actin and glyceraldehyde-3-phosphate dehydrogenase as a pair of genes with the lowest stability value.


CONCLUSIONS
The combination of beta-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, beta-actin and beta2-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended.
Brkljacić
Jelena
J
Department of Biochemistry, Institute for Biological Research Sinisa Stanković, University of Belgrade, 142 Despot Stefan Blvd., Belgrade, 11000, Serbia.
Tanić
Nikola
N
Milutinović
Danijela Vojnović
DV
Elaković
Ivana
I
Jovanović
Sanja Manitasević
SM
Perisić
Tatjana
T
Dundjerski
Jadranka
J
Matić
Gordana
G
D000199:Actins / Q000235:genetics; D020869:Gene Expression Profiling; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008297:Male; D013313:Stress Disorders, Post-Traumatic / Q000235:genetics*; D014728:Veterans
D000199:Actins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases