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Old 02-02-2012, 06:56 AM   #4
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Location: Purdue University, West Lafayette, Indiana

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Originally Posted by Lucilia View Post
Hi Mellissa
I have a worse situation than Nigel. My sequencing results showed 86% of rRNA ( I have to check the parameters used). The service provider showed me the mRNA agilent profile and it indicated 11% of rRNA contamination (poly A purification). How did it become 86%? What percentage of ribosomal RNA contamination is acceptable in the purified mRNA for 454 sequencing?
Probably you were shown an Agilent Bioanalyzer RNA Nano chip run in "mRNA" mode. The analysis must presume that the ribosomal RNA is still intact. If any degradation happened along the way (or was present initially) then rRNA starts to look like just plain old RNA. Hence, not an accurate measurement of the amount of rRNA in the polyA purified fraction.

I don't think there is any standard as to what is "acceptable". Personally I have found that a single cycle of rRNA depletion does a poor job of removing all the rRNA. But yields from doing 2 cycles are terrible usually.

The amount of polyA in your sample will be organism and tissue specific. And this probably plays an enormous role in amount of rRNA left after purification. Actually I mentioned in another thread (some years ago?) a paper where they posed the following thought experiment. Imagine a sample that is 95% rRNA. Say you have a protocol that will remove 90% of rRNA from a sample during a single cycle of depletion. You might naively expect only 10% rRNA to remain after one cycle. But this is mistaken.

What you expect is that 90% of the rRNA is removed. What does that mean in real terms? Think of 100 RNA molecules -- 95 are rRNA and 5 are non-rRNA. 90% of 95 is about 86 molecules of rRNA molecules being removed. That leaves 9 (95-86) molecules of rRNA and the 5 non-rRNA molecules. So your sample is still 65% rRNA (9/16). That is the theoretical best case. So in cases where your polyA RNA makes up a very small fraction of the total RNA, either you do 2 cycles of depletion and your yields suck, or your do 1 cycle and sequence mostly ribosomal.

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