Tru Seq-Very faint bands of small RNA library

[jaymiRNA]

Hi Folks
Iam using Tru seq small RNA prepatation kit for library construction and observe very faint band of small RNA library .
MY ISSUE:
I pooled my 8 plant small RNA libraries and run them on a 6% TBE gel and I observed the desired micro RNA band of 147 and 157 nt are not clear and sharp and they are very faint.
1. I used 2.5ug of small RNA as input material,will using more than 1ug of small RNA as starting material have any problem?.My small RNA sample used for this protocol was intact and not degraded.
2. I tried to run PCR with maximum cycles recommended (15 cycles)
3. Will pooling only 5 ul of each sample with 8 different indexes and loading them on gel result in this faint band/low amount of library?

Please advise me on this

thankyou
Jay

[mikaelk]

I am bit late here but I hope this might still help someone.
The first question would be for me : why so much smallRNA ? or are you talking about totRNA ?
I have been using the small RNA kit for quite a while. Starting with 10µg of totRNA as input before the first gel selection (before lib prep), I never ended with a clean sharp band on the final gel. But cutting in the expected area quite always led to a library when controlling on a Bioanalyzer DNA hi-sens chip (even with quite faint bands).
By the way, I am not working with plants but with a parasite having not much of smallRNA.

I don't know exactly what you are trying to achieve here with your huge amount of RNA but globally, I never ended with really concentrated libraries using smallRNA kits compared to mRNA preps for example. As long as you have a bit more than 2nM of library, I would say you have plenty enough material to sequence without getting troubles with dilutions.

[yangjianhunt]

I know it's late a reply. I hope it might be helpful to others.
For plants, the TRUseq kit seems to be much less efficient than with animal samples. I have used TRUSeq to prep library for both plant and animals, side by side. The difference is very sharp. I started with total RNA, since illumina claims TRUSeq is a gel free system that is compatible with total RNA.
With animal samples (mouse), I always get sharp library bands at around 150bp. With plant samples, there is only a smear in 147-160bp region.

[NextGenSeq]

We increase the cycle number to 18 for small RNA libraries.

[vtosha]

We use only smallRNA as input not total RNA, for plant as for animal. With total RNA as input output libraries too low.
Adapters attaching may be not so good.
And after gel cutting: faint - how much in nM? From 2nM - it's normal.

[jcunnington]

I am also interested in this. Have many people found that you get much better results using TruSeq kit with plant samples when you start with purified small RNA.

I see there were a few posts about a year ago on this. There was talk about comparing with the NEB small RNA kit. I am very interested to know which methods people are using for plant small RNA.

[vtosha]

I think no difference which kit (Illumina or NEB) you use because Illumina get their reagents from NEB (I think so by some reason). We try both kits, on smallRNA of plant, human and fly.
Main difficultiy - how to get smallRNA. If you have smallRNA, then there no differences.

[jpelletier]

We work with plants and have used with success the TruSeq small RNA kit. We found that it works better with total RNA than the small RNA fractionated with the Ambion miRVANA kit. (Much greater % of microRNAs vs other junk, amongst other things.) For us, whether or not we see a sharp ~150bp peak/band depends on the tissue. We never see the 160bp band as plants supposedly do not have the piwi RNAs...

[vtosha]

I got smallRNA with Qiagen Kit. Do you mean % of miRNA in final reads?