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Polysome Analysis

Dipak Kumar Poria1, Partho Sarothi Ray1

  • 1Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India.

Bio-Protocol
|June 13, 2017
PubMed

Insights

Polysome analysis separates actively translating messenger RNAs (mRNAs) from non-translating ones using sucrose density gradient ultracentrifugation. This method helps investigate mRNA translational states by detecting mRNA association with ribosomal fractions.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Understanding mRNA's translational state is crucial for gene expression regulation.
  • Polysome analysis provides insights into which mRNAs are actively translated within a cell.

Purpose of the Study:

  • To describe a protocol for polysome analysis.
  • To differentiate between actively translating and non-translating mRNAs.

Main Methods:

  • Cell lysates are fractionated using sucrose density gradient ultracentrifugation.
  • Fractions containing free mRNA, 40S, 60S ribosomal subunits, monosomes, and polysomes are collected.
  • Reverse transcription followed by PCR (RT-PCR) is used to detect specific mRNA presence in each fraction.

Main Results:

  • The protocol successfully separates various mRNA populations based on their translational activity.
  • Differential association of mRNAs with ribosomal fractions indicates their translational status.

Conclusions:

  • Polysome analysis is an effective method for assessing mRNA translational states.
  • This technique aids in studying the regulation of gene expression at the translational level.
Keywords:
PolysomeRibosome fractionationSucrose density gradientTranslationUltracentrifugationmRNA

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