Brain Research Protocols


Brain Research Protocols 10(2003):156-167


Methods To Optimize The Generation of cDNA From

Postmortem Human Brain Tissue

Christine L. Miller and Robert H. Yolken


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 transcriptase (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.