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Old 06-03-2011, 02:11 AM   #1
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Default Truseq RNA Prep Kit Questions

Hi, hoping to get a few answers/see what others are getting from the Truseq RNA sample kit.

I have just completed several trial runs, from my results using Bioanalyser to qualify success the libraries seem to be ~1/4 of what is shown in the handbook, thankfully they are ~260bp. So I am confident the protocol has worked, but am now wondering what to do to increase yield.

Does anyone use any modifications to the standard procedure? I have been thinking:
(i) increase in PCR enrichment cycles; anecdotally the PCR enrichment step does not suffer from PCR duplicates: is this really the case?
(ii) use low-bind plates; I have used what we had in the lab but should probably find some low-bind plates: has anyone found this to make a difference in yield? Any recommendations for plates?
(iii) is it because my RNA is not good quality? RINs ~8 which seems to be acceptable. Was going to try with some very high quality RNA but wanted to know others experience. Surely if it is fragmented during lib. prep. it will not matter so much about the RNA integrity if it isn't badly degraded(?)

I will sequence and run qPCR but would like advice about issues of yield. First time doing libraries and the lab I am in has a DIY which has worked very well so not sure whether to go along with Truseq or just do the longer (but working) lab method.

Thanks in advance,

bruce01 is offline   Reply With Quote
Old 06-07-2011, 01:28 PM   #2
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Hi Bruce,
(i) If you need more library to fill out your run -- getting that extra library by increasing enrichment cycles probably will result in additional PCR duplicates. Stands to reason...
(ii) Depends on the binding capacity of normal plates. I don't know what that is. Maybe 1 ng?
(iii) Yes. The TruSeq RNA kit entails a polyA RNA binding. If your RNA is degraded, then only that RNA adjacent to the polyA tail will be retained. The 5' ends of the RNA strands will be lost. That, among other things, will lower yield.

pmiguel is offline   Reply With Quote

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