Truseq small RNA library:138 and 140 bp peaks after gel purification

[shahideh]

Hi guys,

I made two small RNA libraries by using Truseq kit. I am looking for any kind of small RNA to identify any kind of virus in these two populations. After gel purification, I run libraries on chips from Bio-Rad (Experion). It seems that I have those expected sizes (147, 149, 154) but I also have 138 and 140 bp peaks (Attached). The last run is just blank! I was wondering if they are adapter dimers. I am still confused with 7 bp shifting of libraries on gel and bioanalyzer which illumina talks about that. Do you think it is better to make new libraries?

Thanks,

[kcchan]

Do you have any leftover PCR product that you can run the bioanalyzer on? If you overlay the two traces you should be able to tell if your peaks are small RNA or adapter dimers. It is possible that either the bioanalyzer is off by a few bases or your gel cut could be slightly off.

[shahideh]

Thanks Kcchan for your reply. I have already run the PCR products before gel purification (Attached). I pooled sample 1, 2 and 4 and run on a gel. So, sample 1 from after gel purification is the result of mixed samples 1, 2 and 4 of before purification. Sample 3 from before purification trace is equal to sample 2 from after purification trace. Sorry for confusion

[kcchan]

The adapter dimer peak looks very high in your PCR products. It's likely that your gel cut wasn't precise enough and the dimers still remain in your gel purified libraries. I would suggest repeating the experiment if you still have RNA that you can use.

Is there anything different about this set of samples compared with your previous set? It is a bit unusual to see the adapter dimer peaks be that intense.

[shahideh]

The only difference was input RNA. I used total RNA to make my previous library which was perfect. This time I used small RNA extracted by using mirVana kit to start making library. I used 400-500 ng sRNA but maybe it was low!!!! Of course I looked the traces of my previous library before gel purification. It is very similar to the new library. But after gel purification I could get ride of that completely!

Again thanks

[kcchan]

400-500ng of pure small RNA is more than enough for library generation. We've tried amounts as low as 10ng and were successful. You repeat the experiment again, making sure the gel cut is at the right place. Since the adapter dimers are fairly high, it may be difficult to distinctly see the 136 and 147 bands. The included custom ladder provides a good indicator of where the cut should be made.

[shahideh]

I agree with you. I should repeat it! I have attached the gel after cutting the band. I tried to cut the area btw two bands of custom ladder but as you said it is very hard to get ride of 136 bp band.

[shahideh]

Oops! Sorry I forgot to attache. Here you go