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 tran script (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 care fully 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 13 actin had lower overall expression in AD and AD/LBD cases than in controls RPL13 and 18S were the most stably expressed house keepers tested Synaptophysin and ghat 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 (C) 2010 The Alzheimer's Association All rights reserved