Methods to optimize the generation of cDNA from postmortem human brain tissue.
The analysis of gene transcript levels in postmortem human brain is a valuable tool for the study of neurological and psychiatric diseases. Optimization of the methods of RNA extraction and cDNA generation is particularly important in this application because postmortem human brain tissue is in limited supply and generally yields less RNA than many other human tissues. We compared column extraction and solvent extraction for total RNA, reverse transcription (RT) with random hexamers versus oligo-dT priming, and incubation of the RNA with or without DNase for effect on the cDNA product derived from the same homogenized pool of postmortem human frontal cortex. The total RNA obtained from the solvent method was found to be less stable at room temperature and to contain a higher proportion of non-messenger RNA than that obtained from the column method. Evaluating the RT-PCR results per wet weight of tissue extracted, we found that the signal strength was increased >20-fold by a protocol of Qiagen RNeasy column extraction, random hexamer RT priming and omitting DNase treatment of the RNA.
Miller
Christine L
CL
Stanley Division of Developmental Neurovirology, Department of Pediatrics, Johns Hopkins University School of Medicine, 600 N. Wolfe St./Blalock 1105, Baltimore, MD 21287, USA. cmiller2@jhmi.edu
Yolken
Robert H
RH
D001923:Brain Chemistry; D018076:DNA, Complementary / Q000302:isolation & purification*; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007202:Indicators and Reagents; D012333:RNA, Messenger / Q000302:isolation & purification; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000737:chemistry; D013696:Temperature
D018076:DNA, Complementary; D007202:Indicators and Reagents; D012333:RNA, Messenger; D012260:Ribonucleases; D003850:Deoxyribonuclease I