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.
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References
Byers RJ, Hoyland JA, Dixon J, Freemont AJ (2000) Subtractive hybridization-genetic takeaways and the search for meaning. Int J Exp Pathol 81:391–404
Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971
Liang P, Pardee AB (1995) Recent advances in differential display. Curr Protoc Mol Biol 7:274–280
Huang Z, Fasco MJ, Kaminsky LS (1996) Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR. BioTechniques 20:1012–1020
Jones SW, Cai D, Weislow OS, Babak EA (1997) Generation of multiple mRNA fingerprints using fluorescence-based differential display and automated DNA sequencer. BioTechniques 22:536–543
Zhao S, Ooi SL, Pardee AB (1995) New primer strategy improves precision of differential display. BioTechniques 18:842–850
Doss RP (1996) Differential display without radioactivity—a modified procedure. BioTechniques 21:408–412
Pfeffer U, Fecarotta E, Vidali G (1995) Efficient one-tube RT-PCR amplification of rare transcripts using short sequence-specific reverse transcription primers. BioTechniques 18:204–205
Averboukh L, Douglas SA, Lowe K, Maher J, Pardee AB (1996) Better gel resolution and longer cDNAs increase the precision of differential display. BioTechniques 20:918–921
Chen JJW, Peck K (1996) Non-radioisotopic differential display method to directly visualize and amplify differential bands on nylon membrane. Nucleic Acids Res 24:793–794
Callard D, Lescure B, Mazzolini L (1994) A method for the elimination of false positives generated by the mRNA differential display technique. BioTechniques 16:1096–1103
Biswas P, Majumdar U, Ghosh S (2015) Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display. Data Brief 6:101–111
Majumdar U, Biswas P, Subhra Sarkar T, Maiti D, Ghosh S (2012) Regulation of cell cycle and stress responses under nitrosative stress in Schizosaccharomyces pombe. Free Radic Biol Med 52:2186–2200
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔC T method. Methods 25:402–408
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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Biswas, P., Majumdar, U., Ghosh, S. (2018). Analysis of Reverse Transcribed mRNA Using PCR and Polyacrylamide Gel Electrophoresis. In: Singleton, T. (eds) Schizosaccharomyces pombe. Methods in Molecular Biology, vol 1721. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7546-4_7
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DOI: https://doi.org/10.1007/978-1-4939-7546-4_7
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