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Old 09-05-2011, 05:36 AM   #1
eoh001
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Location: seoul Korea

Join Date: Mar 2011
Posts: 19
Unhappy mRNA extraction from frozen tissue

Hello
I am planning to sequence the transcriptome of various frozen tissues using SOLiD4. According to the 'SOLiD Total RNA-Seq Kit protocol', two rounds of poly-A selection(MicroPoly(A)Purist Kit) are recommended to prepare RNA samples for library construction. However, the concentration of the poly(A) RNA at the final step was pretty low, and even undetectable in certain tissues. I wonder whether I did something wrong or it was a general case. Since I have limited amount of the tissues, I hope to get any idea to increase the mRNA yield.


1. I extracted the total RNA from the frozen tissues using the relevant Quiagen Kit, and selected poly-A RNA following the MicroPoly(A)Purist Procedure. The RIN of total RNA was around 9-10, the ratio 28S:18S was OK, and the amount of total RNA was good enough. However, the concentrations of poly-A RNA were way lower than expected (less than 1% of total RNA, about .5%). I had to increas the concentration of poly-A RNA in the RNA storage solution by ethanol precipitation. The problem was that the amount of poly-A RNA reduced by half after ethanol precipitation. Getting worse (total RNA x 0.5% x 50%= total RNA x 0.25%). If I perform two rounds of poly-A selection, it would be extremly low.
I was told that freezing dry would be better than ethanol precipitation in terms of yield. Is it true? Or any better way to minimize the loss of poly-A RNA?

2. If the method I am using now (2 times of poly-A RNA selection) is not a good choice,
Which one is better in my case, (1) poly-A RNA selection(1 time) + Normalization kit (such as DSN) (2) total RNA+RiboMius (3) total RNA + Normalization kit.


I'll really appreciate your help!!!
Thank you.
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Old 09-06-2011, 05:15 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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If yield is low after one cycle of ribo depletion, we skip the second cycle. Usually rRNA is brought below 30% or so with a single cycle.

We also generally see poly A yields below 1%.

The Ambion/LifeTech Total RNAseq lib prep method, for all its charms, does ask for a vast amount of ribo-depleted RNA to start with. 200 ng of RNA is roughly 800 billion 500 nt RNA fragments.

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