Housekeepers for accurate transcript expression analysis in Alzheimer's disease autopsy brain tissue.
BACKGROUND
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a popular technique for mRNA expression studies. Normalization to an endogenous reference transcript (housekeeper) is widely used to correct for differences in loading and RNA quality. Alzheimer's disease (AD) alters brain metabolism. The stability of housekeeper transcript expression must be carefully validated.


METHODS
qRT-PCR was used to assess eight putative housekeeper transcripts in four brain regions from 15 control, 12 AD, and 10 AD/Lewy body disease (LBD) cases.


RESULTS
RNA quality is lower in AD and AD/LBD than in controls. Frequently used housekeepers such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin had lower overall expression in AD and AD/LBD cases than in controls. RPL13 and 18S were the most stably expressed housekeepers tested. Synaptophysin and glial fibrillary acidic protein were used to evaluate normalized quantification. By using different housekeepers we confirmed that synaptophysin expression was down-regulated in AD cases, whereas glial fibrillary acidic protein expression was increased.


CONCLUSIONS
Among all candidates tested, RPL13 was the best housekeeper for qRT-PCR studies in autopsy brain tissue samples from controls and AD cases. RNA quality should be assessed and data normalized on this index as well.
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia.
Florian M
FM
Gebhardt
Heather A
HA
Scott
Peter R
PR
Dodd
D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D001344:Autopsy / Q000379:methods* / Q000592:standards; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D001923:Brain Chemistry / Q000235:genetics*; D005260:Female; D005819:Genetic Markers / Q000235:genetics*; D006801:Humans; D008297:Male; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000592:standards*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000592:standards; D012015:Reference Standards; D012016:Reference Values; D014158:Transcription, Genetic / Q000502:physiology*
D015415:Biomarkers; D005819:Genetic Markers; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger