mRNA enrichment (View forum version)


Posted 27 April 2010 - 01:40 AM

Can anyone tell me how I enrich for a mRNA fraction? I am doing an extraction RNA, then converting to cDNA. I am going to use a the first couple of steps of the exactSTART kit to isolate full length cDNA using the TAP enzyme to tag the 5' end(ie complete 5' sequences) and have been told I should enrich for the mRNA fraction.


Posted 27 April 2010 - 04:37 AM

If you are working in a eukaryote, then there are enzyme mixes (also from Epicentre) that will degrade uncapped RNA. You can also capture rRNA (the other major species) with beads containing the complementary sequences. I believe Ambion (now Life Technology) sells these, as well as several other suppliers.


Posted 27 April 2010 - 01:40 PM

As phage434 said, you can use the mRNA-ONLY kit to degrade uncapped RNA. This is a relatively quick method and has been used in combination with the ExactSTART kit for preparing cDNA libraries for 454 sequencing (see doi:10.1038/nmeth.f.257).