Methods to optimize the generation of cDNA from postmortem human brain tissue

Christine L. Miller, Robert H Yolken

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)156-167
Number of pages12
JournalBrain Research Protocols
Volume10
Issue number3
DOIs
StatePublished - Feb 2003

Fingerprint

Complementary DNA
RNA
Brain
Reverse Transcription
Deoxyribonucleases
Frontal Lobe
Psychiatry
Weights and Measures
Polymerase Chain Reaction
Temperature
Genes

Keywords

  • Column
  • DNase
  • Human brain
  • RNA extraction
  • RT priming
  • Solvent

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Methods to optimize the generation of cDNA from postmortem human brain tissue. / Miller, Christine L.; Yolken, Robert H.

In: Brain Research Protocols, Vol. 10, No. 3, 02.2003, p. 156-167.

Research output: Contribution to journalArticle

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