Problems getting results using Illumina / NEB kit for small RNA library prep.

[Martin Mikkelsen]

Dear all,

Does anyone have any experience with these kits?

I was able to make one successful build using the Illumina kit and since then has everything failed. I have also tried the similar kit from NEB.

I have used Ambion brain, liver and testes including some internal in-house controls containing known amounts of small RNA species.

How stable are the reagents in the kit?
Should I worry about my samples? I have used the recommend 1 µg input material for both kits. And QC-ed my samples using the BioAnalyzer RNA kit.

Hope that someone has an idea of what could be wrong or how to proceed!

[Martin Mikkelsen]

I solved the problem (well somebody in the lab helped) me.

We used heating in thermocycler lid. Somehow it affected the incubations steps. Now we dropped the temperature in the lid and everything seems to work.