SMARTer Stranded Total RNA kit

[GTatiana]

We are working with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara). When we prepared libraries for the first time, results was acceptable – we get libraries from control RNA (total input amount 0,25 ng/μl ) and from LCM tissue (track A-C in attached file) with one exception – low yield (approx. 0,8 ng/μl in all samples). However, all our next attempts to create libraries were unsuccessful and we cannot find the reason. We took into account all recommendations about sample storage, preventing contamination and all general requirements. Before work with RNA we treated workflow with RNaseZap, before work with cDNA – DNAZap. Kit didn’t work even when we used only control RNA with different input amount (0,75 ng/μl, 10 ng/μl and even 20 ng/μl). In attached file, I demonstrate TapeStation electrophoresis from all stages of our experiment. Seems to be, problems appear in the beginning of protocol, after stage of first-strand cDNA synthesis and PCR1 (where is no necessary amplified products on the electrophoresis).
Is anybody face with the same problem?

https://drive.google.com/file/d/1sx0B0daXE8Qq7P-BHCFaKgjjpiXIOgtV/view?usp=sharing

Many thanks

[JamesHrastelj]

?Over-dried AMPure beads?

Hi,

I have been using the same library prep kit. Although it's difficult to be sure exactly what has caused your problem, I know that one of the most sensitive parts of the protocol is the AMPure bead purification, so close attention should be paid to getting that part exactly correct. For example, if the bead pellet becomes overdried then the yield is significantly affected. The pellet should be covered with RNase free water as soon as the very first tiny crack appears in the surface of the pellet.

The other possibility that springs to mind is RNase contaminated reagents (e.g. water).

Good luck!

[Dorota]

Is it possible that your original RNA sample is degraded to begin with ? Or was the new control material a different RNA with low likelihood of being degraded?