Validation of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics.
Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large P0 (RPLP0) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes.
Johansson
Sofia
S
Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. Sofia.Johansson@ki.se
Fuchs
Andrea
A
Okvist
Anna
A
Karimi
Mohsen
M
Harper
Clive
C
Garrick
Therese
T
Sheedy
Donna
D
Hurd
Yasmin
Y
Bakalkin
Georgy
G
Ekström
Tomas J
TJ
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D020268:Alcohol-Induced Disorders, Nervous System / Q000235:genetics*; D000437:Alcoholism / Q000235:genetics*; D001284:Atrophy / Q000139:chemically induced / Q000235:genetics / Q000503:physiopathology; D002492:Central Nervous System Depressants / Q000009:adverse effects; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism / Q000503:physiopathology; D000431:Ethanol / Q000009:adverse effects; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009044:Motor Cortex / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology; D009419:Nerve Tissue Proteins / Q000187:drug effects* / Q000235:genetics*; D017397:Prefrontal Cortex / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D015203:Reproducibility of Results; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D002492:Central Nervous System Depressants; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D000431:Ethanol