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Old 11-19-2014, 02:29 AM   #12
mikaelk
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Location: paris

Join Date: Jun 2012
Posts: 11
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Hi,

I would recommend to gel purify your samples before starting the library construction AND after the PCR. I used for the first gel a ladder called ZR small RNA ladder from Zymo to correctly evaluate the size of miRNAs as for example the Illumina ladder were having a shift of 10 bases in my hands.

It is indeed longer but by doing so, I have been able to get really clean and good libraries.

I have been constructing libraries for some years now and one thing that is always true is the classical GIGO, garbage in --> garbage out. Either you filter directly when you construct the library or you sequence garbage that you have to filter bioinformatically.

The differences in size between the different small RNA populations is so small that you will always get contamination from other fractions but the gels really decrease the problem.

The size selection should be rather around 140 bp as indicated before. Adpater dimer being around 120bp, if you size select at 155bp, it means you have and insert size of 35bp that do not correspond to miRNAs...

In the best case scenario, one should size select first between 18 and 30nt on 15% TBE UREA polyacylamide gel and then purify the libraries on a 5 % TBE polyacrylamide gel selecting between 135 and 150 bp to get a clean result.
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