Semi-quantitative reverse-transcriptase polymerase chain reaction: An approach for the measurement of target gene expression in human brain

Li Chen, David M. Segal, Deborah C. Mash

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

Polymerase chain reaction (PCR) is a very powerful tool for qualitative evaluation of nucleic acids due to its high efficiency and convenience. Together with the reverse transcription (RT) reaction, the PCR method has been widely applied to the quantitative measurement of DNA and RNA messages. Since RT-PCR is much more sensitive than all of the traditional methods for quantification of mRNA, including Northern blot, ribonuclease protection, RNA blot, and solution hybridization assays, it is the method of choice for quantitative analyses of low abundance mRNA messages. However, because of the exponential nature of the PCR amplification, RT-PCR quantitation may be problematic, giving false estimates of the abundance of the target messages. By using the constitutively expressed 'housekeeping' gene cyclophilin as a reference gene to normalize mRNA levels, and by taking data from the exponential phase of the PCR amplification, we have developed a rapid and reliable semi-quantitative measurement of the relative abundance of dopamine transporter (DAT) mRNA. The semi-quantitative PCR method has been applied to illustrate its use for the measurement of DAT mRNA in post mortem human brain.

Original languageEnglish (US)
Pages (from-to)132-139
Number of pages8
JournalBrain Research Protocols
Volume4
Issue number2
DOIs
StatePublished - Jul 1 1999

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Keywords

  • Dopamine transporter (DAT)
  • Human brain
  • Polymerase chain reaction (PCR)
  • Reverse transcription (RT)

ASJC Scopus subject areas

  • Neuroscience(all)

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