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Analysis of Reverse Transcribed mRNA Using PCR and Polyacrylamide Gel Electrophoresis

  • Pranjal Biswas
  • Uddalak Majumdar
  • Sanjay GhoshEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1721)

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.

Key words

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

Notes

Acknowledgments

The consumables for the entire work were supported by DBT, Govt. of India, grant No. BT/PR12551/BRB/10/02/2009 dated September 3, 2010 to SG, DBT-IPLS, UPE, DST-FIST, Indian Council of Agricultural Research (ICAR), Govt. of India and UGC CAS Phase II Govt. of India for providing infrastructural facility. CSIR, Govt. of India for providing fellowship to PB and DBT, Govt. of India for providing fellowship to U.M.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Pranjal Biswas
    • 1
    • 3
  • Uddalak Majumdar
    • 2
    • 3
  • Sanjay Ghosh
    • 3
    Email author
  1. 1.Department of Genetics, Cell Biology and AnatomyUniversity of Nebraska Medical CenterOmahaUSA
  2. 2.Center for Cardiovascular ResearchNationwide Children’s HospitalColumbusUSA
  3. 3.Department of BiochemistryUniversity of CalcuttaKolkataIndia

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