Truseq RNA Prep Kit Questions

[bruce01]

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,

Bruce.

[pmiguel]

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.

--
Phillip

[bruce01]

Thanks for your reply Phillip,

yes, it does stand to reason that PCR dupes are created with additional cycles, for our DIY method we have reduced to 7 or 8 cycles as this is a big problem. I have heard from technicians that dupes are reduced in the Illumina kit but have not seen any proof of this (yet!)

I am going to try a sample with very high RIN and 28/16 ratio to determine if degradation plays a major part in my lower yield. I can see how degradation at 5' would affect yield, just hoping the method isn't very sensitive to this.

Illumina techsupport have suggested a vacuum concentrator or preparing more library and pooling, but I am not too keen as we are cutting it fine with number of preps in the kit as it is.

Bruce.

[bruce01]

Hi Phillip,

thanks for the reply. Was waiting to test a few more samples prior to replying myself.

(i) Illumina techsupport suggest: making another library to add to the previous one to boost yield; use a vacuum concentrator (without heat). Agree with increasing PCR cycles, do not want to bias more than we already do.

(ii+iii) constructed a very nice library (akin to the image in the handbook) using a very good quality RNA sample (RIN 9.5, 28/18 2.1). Also left XP steps for longer to increase binding which may have helped.

There is a 'bump' after the ~260bp library, ~550-2000bp which I assume is insert/adapter concatenators(?)

Cheers,

Bruce.