RNA Seq Low Yield of Libraries

[SciConnect]

TruSeq libraries have been generated, but are extremly low (about 0.3 ng/uL). I usually save half of my ligated product in case I need to go back to it. Is it possible to re-do A-tail step, and ligation of adapters, and then to PCR again, then pool with low original libraries.

Its seems as if ligation of adapters were not as efficient.

[kerplunk412]

Hi Sci, I think your plan sounds good, except that if you are successful I don't think there should be any need to pool the new libraries with the old ones. Also, if there is an end repair step in your protocol you may want to repeat that as well.

[Manna]

With the TruSeq kit, I typically finish with 30 µL of library at a concentration of around 10-20 ng/µl, sometimes as much as 40. Anything less than 5 ng/µl is suspect, meaning, it isn't likely to sequence very well if at all. Try running your library on a bioanalyzer and see if you get anything. You can also take a small bit, like 1 µL, and see if you can PCR it up and get more. If you can't, your library is no good.

The most typical reason for an mRNA library prep to fail is lousy starting material.

**I've literally made over 1,000 mRNA TruSeq libraries.

[NewbieNGS]

We had the same problem in November and it turned out to be related to a performance issue with the PCR Master Mix reagent found in our kit. Illumina replaced the kits but we lost our samples.

http://www.illumina.com/index-d.html...bFLM14380QUhPl

[mayankkaashyap]

I have used TruSeq Total RNA kit and have got final libray conc. 2.22 ng/ul. Is it ok to proceed for sequencing.

[luc]

Do you have bioanalyzer traces?

I have used TruSeq Total RNA kit and have got final libray conc. 2.22 ng/ul. Is it ok to proceed for sequencing.