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Total RNA Isolation and Quantification of Specific RNAs in Fission Yeast

  • Robert Roth
  • Hiten D. Madhani
  • Jennifer F. GarciaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1721)

Abstract

The fission yeast, Schizosaccharomyces pombe, is an important model organism for investigations of gene regulation. Essential to such studies is the ability to quantify the levels of a specific RNA. We describe a protocol for the isolation and quantification of RNA in S. pombe using reverse-transcription followed by quantitative PCR. In this procedure, the cells are lysed using zirconia beads, then total RNA is selectively isolated away from proteins and DNA using the Trizol reagent. Contaminating DNA is then removed from the RNA by using TURBO DNase, which is easily inactivated and requires no subsequent clean-up step. The RNA is then reverse transcribed into cDNA using random nine-mers and oligo dT primers. Quantitative PCR using SYBR green is then performed to quantify RNA levels. This protocol has been tested on several S. pombe genotypes and generates highly reproducible results.

Key words

RNA extraction Reverse-transcription qPCR cDNA synthesis DNase RNase RNA quantification SYBR green 

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Robert Roth
    • 1
  • Hiten D. Madhani
    • 2
  • Jennifer F. Garcia
    • 1
    Email author
  1. 1.Department of Molecular BiologyColorado CollegeColorado SpringsUSA
  2. 2.Deptartment of Biochemistry and BiophysicsUniversity of CaliforniaSan FranciscoUSA

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