Analysis of reverse transcribed mRNA using PCR and polyacrylamide gel electrophoresis

Pranjal Biswas, Uddalak Majumdar, Sanjay Ghosh

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The patterns of gene expression in the fission yeast Schizosaccharomyces pombe under various experimental conditions form the basis of any transcriptomic study. We describe a method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast. The method described is cost effective and reproducible with satisfactory resolution of expressed transcripts in the gel. The method has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, re-amplification and cloning of the differentially expressed PCR products, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. The technique is also popularly known as Differential Display Reverse Transcription (DDRT-PCR). After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of “false positives.” Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of biology, DDRT-PCR has become a very popular molecular technique during the past two decades.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages73-87
Number of pages15
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1721
ISSN (Print)1064-3745

Keywords

  • Differential display
  • Polymerase chain reaction
  • Reverse transcription
  • Schizosaccharomyces pombe
  • Urea-polyacrylamide gel
  • cDNA
  • mRNA

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Biswas, P., Majumdar, U., & Ghosh, S. (2018). Analysis of reverse transcribed mRNA using PCR and polyacrylamide gel electrophoresis. In Methods in Molecular Biology (pp. 73-87). (Methods in Molecular Biology; Vol. 1721). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7546-4_7