Small RNA-Seq Kits

[l_zembek]

I need to prepare some miRNA libraries for Illumina sequencing, and have been looking at what's available. Does anyone have any experience with any of these kits, good or bad?

-TruSeq Small RNA Sample Prep Kit
-Ambion® RNA-Seq Library Construction Kit
-ScriptMiner™ Small RNA-Seq Library Preparation Kit (EpiCentre)
-NEXTflex™ Small RNA Sequencing Kit (Bioo Scientific)

Also, all the kits seem to require a gel cut following cDNA synthesis. Has anyone used a PippinPrep or SizeSelect gel in lieu of a gel cut? I just hate gel cuts...

Thank you!!!

[garciad1]

I have done quite a few small RNA library preps. We tried the TruSeq kit first, but didn't catch that you have to supply one of the ligases and used the one in the kit for both ligation steps. I now routinely use the BIOO kit followed by an acrylamide gel size selection. We have a LabchipXT rather than a pipen prep and were unsuccessful at using that in place of the acrylamide gel (I hate isolating from a gel as well). Unfortunately we always got some primer dimer carry over. They have since come out with a technote on how to use the labchipXT for this type of library but since I'm getting good results with the acrylamide gel I haven't revisited using the labchip.

[NextGenSeq]

Note all of these show severe bias against some miRNAs due to ligase bias. Due a quick PubMed search.

[l_zembek]

Thank you for your comments! I think I'll try the PippinPrep, since we have one, and have had good results at isolating bands in that size range.

It does appear that using adapters with randomized regions will result in higher ligation efficiency, thank you.

[Genohub]

Small RNA Seq Bias

This article nicely describes the bias issues with Small RNA Seq

Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing

[SageSigh]

http://www.epibio.com/applications/rna-sequencing/rna-library-prep/pippin-prep-for-scriptseq-

Apologies for the (shameless) self promotion (I work for Sage Science), but the above link provides Pippin Prep data for RNA-seq and introduces Epicentre's new kit. I am curious to know how it compares to other RNA products on the market. Has anyone tried the Epicentre product?

[Biochain Rep]

I would also like to shamelessly plug our small RNAseq kit (please see www.biochain.com) as an alternative. We include virtually all required materials, including cutters for the acrylamide gel.

Now that my plug is done... I'd really like to know how the Pippin Prep would work out as a replacement for the gel isolation step. I'm thinking that that this device would make the process a lot more convenient. Not show what the costs would be...