Problem in Making Small RNA Library from Grape

[sunitha]

Hello All

I am having trouble in amplifying small RNA library using Tru-Seq small RNA kit. I used 1 ug of Total RNA from grape leaf. I had checked the quality using Bioanalyzer (RIN was >8). I have followed the steps as mentioned in the manual precisely. I repeated twice with no result. Please help me trouble shoot this issue.

[nucacidhunter]

Failure could be due to lack of small RNA in the total RNA prep. Extraction method may have not conserved small RNA fraction.You can rule out this possible cause by running samples on Small RNA Chip.

[sunitha]

Thank you for your reply. I suspected the same and probed the total RNA with miR166 and found there is accumulation of small RNA but relatively small amounts. I will take your advise and run a small RNA chip. I am also trying to enrich small RNA by miRPremier microRNA purification kit (sigma). Tru Seq recommends 50 ng of small RNA for library preparation. I hope enriching small RNA will resolve my issue.

[sunitha]

Hello All

I tried making small RNA library using small RNA purified using miR Premier kit. I did not succeed in getting amplification. We do not have small RNA chips to do bioanalyzer test. Please let me know your suggestions to overcome this issue.

[nucacidhunter]

Using total RNA with 10 ng miRNA content or 10 ng of purified small RNA works well with standard TruSeq small RNA library prep protocol. The input can be as low as 2 ng miRNA in which case PCR cycles need to be increased to compensate for lower input. I would suggest following:

1- If you do not access to small RNA Chip, you can check success of miRNA extraction on gel as described in the kit user guide.
2- It is important that RNA is resuspended in the buffer recommended in TruSeq guide.
3- Using Human Brain Total RNA along with your samples as a positive input control for troubleshooting.

[sunitha]

Thank you for your reply. I probed the small RNA purified using miRPremier kit with miR166 probe. I did see hybridization. I had eluted the small RNA in nuclease free water. Right now we do not have human brain sample to use as positive control. Is there any chance that some component in the kit might be causing this issue. How stable are these components upon repeated thawing (on ice) and freezing and centrifuging?

[Olaf Blue]

Grape leaves (Vinifera) typically have issues with complex carbohydrates and polyphenols in the tissues. Possible that these are issues for you? Sometimes, using 1% polyvinylpyrrolidone in the RNA extraction can help.