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. 2017 Mar 20;7(6):e2192.
doi: 10.21769/BioProtoc.2192.

Polysome Analysis

Dipak Kumar Poria  1 , Partho Sarothi Ray  1
Affiliations

Polysome Analysis

Dipak Kumar Poria et al. Bio Protoc. .

Abstract

Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription - PCR to investigate the translational state of the mRNA.

Keywords: Polysome; Ribosome fractionation; Sucrose density gradient; Translation; Ultracentrifugation; mRNA.

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Figures

Video 1.
Video 1.. Gradient preparation
Figure 1.
Figure 1.. 10%-50% sucrose gradient showing 2 mm space on top of gradient
Figure 2.
Figure 2.. Ribosomal fractions from MCF7 cells were analysed by 10-50% sucrose density gradient fractionation.
A. Ribosomal RNA content, measured at 254 nm, is plotted against fraction numbers (top panel). B. RNA isolated from selected fractions was analyzed by semiquantitative RT-PCR using PDCD4 and GAPDH primers (bottom panel).
See this image and copyright information in PMC

References

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