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: Translation, Ribosome fractionation, Polysome, mRNA, Sucrose density gradient, Ultracentrifugation
Background
The cellular mRNAs are distributed into an actively translating and a non-translating pool at any point of time and can dynamically redistribute between these pools in response to various stimuli. The actively translating mRNAs have a higher number of ribosomes associated with them and the number of ribosome associated with an mRNA is a measure of the translation state of the mRNA. Therefore on fractionating the ribosomes from a cell, actively translating mRNAs will be found in the polysomal fraction whereas non-translating/poorly-translating mRNAs will be either found in the free mRNA fraction or associated with 40S ribosomal subunits. Polysome analysis is therefore a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes (Ray et al., 2009; Poria et al., 2016). Association of individual mRNAs with translating/non-translating fractions can be detected by RT-PCR, whereas the entire translation or non-translating pool of mRNAs can be identified by RNA sequencing or microarray analysis.
Materials and Reagents
Equipment
Software
Procedure
Recipes
Acknowledgments
We thank members of our laboratory for trying out and standardizing this protocol. Research which led to the development of these protocols was funded by a Wellcome Trust-DBT India Alliance Intermediate fellowship (WT500139/Z/09/Z) to PSR and a CSIR, India Senior Research Fellowship to DKP. This protocol is modified from the protocol described in Merrick and Hensold (2001).
References
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Hello Runlai, DNase digestion is not necessary for the RNA before RT reaction as cytoplasmic fraction is used for the polysome analysis in this protocol. Cell lysate preparation step in this protocol removes nucleus from the cytoplasmic fraction. Best, Dipak