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Old 12-16-2014, 04:17 PM   #4
kerplunk412
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Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
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mRNA is only 1-3% of total RNA, so as others have said you can't really see it at all in total RNA. Having intact rRNA peaks is basically an indication that you are not seeing significant degradation in your sample, so you can safely assume that your mRNAs are also intact.

I will also save you some confusion later: after rRNA depletion you have a large peak of tRNA, so you STILL can't see the mRNA. Also, since so much of the rRNA depleted sample is tRNA, you will need to use more rRNA depleted sample vs poly(A) enriched sample for library prep. For example, if your protocol calls for 1 ng of poly(A) RNA, you should probably use at lease 10 ng of rRNA depleted RNA.

Also, since you are performing rRNA depletion it doesn't matter much if your RNA is somewhat degraded, as long as it isn't very badly degraded. So I think you can confidently proceed with the RNA you have. Good luck!
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