small RNA Lib, strange bands, need help

[chen]

hi, I havre constructed the small RNA library following the exact protocol of ILMN TruSeq small RNA with ILMN small RNA kit. and excised the bands between 145bp and 160bp, but the agilent 2200 showed the library was only 135bp!(see the attachment), and so as other samples!All samples were human samples.

Did I cut the wrong band? I already try the upper band(B band), and the 2200 result was 172bp. Which band is the right library. This was the first trial experiment in the lab, and we really need help before running.

Thanks in advance!
Tanya

[Genohub]

Small RNA Bands

What is your starting material, total RNA or small RNA isolated from total RNA? How much are you starting with?

Small RNA lp is one of the more difficult library preparations out there. You have to be careful that your excised bands do not contain traces of adapter-dimer. For Small RNA libraries these are around ~120bp and result when the 5'adapter ligates to your 3'adapter and is subsequently amplified. Your gel image indicates a long smear. A smear like that suggests that things could be running together, potentially resulting in dimer contamination in your final results. I suspect the 135bp band is your dimer band. The 172bp band may be the 160bp product running a little high. I don't see that band on the TapeStation. The only way to know for sure (without putting it on a Miseq) is to clone it into a vector or sanger sequence the PCR products and look for small RNAs.

[chen]

hi Genohub,
Thank you very much for your quick reply!

We used 1ug of total RNA of the human sample, and followed the exact protocol of TruSeq small RNA kit(did not isolate samll RNA from total RNA). And yes, we are also considering sending the both library(135bp band and 172bp band) to sanger sequencing. Hope we get result soon.

But still the problem is there. Does anyone get the same result that I did? Or which step may I possiblely do wrong, or should I pay more attention to avoid this problem?

[chen]

we also try agarose gel electrophoresis on the final step, and cut the right size band(~150bp), and still the 2200 result showed ~130bp. Maybe it's just the deviation of the instrument(like ~20bp deviation? not so sure)

[Genohub]

Hi Tanya,

It does look like you are getting a 20bp differential between agarose gel and tape station. Are you using SYBR or EtBr? I'd be curious to see if anyone else sees the same differential on a double stranded PCR product. In the end the Sanger sequencing will tell you whether its a spurious band or true small RNA library.

[chen]

hi Genohub,

we finally got the sanger sequencing of the library(the lower band) and the upper band, and yes, the library(lower band) is right one(small RNA). the upper band is more like the non-coding RNA library.

About the staining, we use SYBR Gold for both agarose gel and the PAGE. Maybe that's the cause? Anyway, we finally got the right library.

[Genohub]

Your SYBR Gold staining is probably fine. You're just getting a slight variation between tapestation and gel. Was this a one time occurrence or do you often notice that gel and your tapestation are off?

[vivian914]

I have the same result that you did, excised the bands between 145bp and 160bp, agilent 2100 showed was around 140bp, after sequencing,the library is right one ,unfortunately,50% of date is ribosome, the RIN of input RNA>9,the lib prep process is the same to the protocol,I don't know why

[Genohub]

If you're getting a lot of ribosomal reads I would recommend isolating the small RNA from your total RNA before starting library prep. That should help. I would also run your final PCR product on the gel longer so that the ribosomal bands have time to separate from your small library.

- Genohub

[Petrinni]

Hi everyone,
we did the same experiment today and we have similar problem. We have used 1 ug of total RNA and we want to sequence miRNAs, but our band on PAGE gel and also on Bioanalyzer was only 75-80bp long. Do you have idea, where the problem could be and what we have gained? Thanks a lot.

Petra