mRNA fragmentation/weird Bioanalyzer profile

[Irigoyen]

Hello everybody
I'm going to prepare RNAseq libraries from cassava. First I did was purify mRNA from my samples using polydT(25) beads (life technologies). Then using 5x First Strand Buffer (Invitrogen) I incubated the samples at 94C for different times (2, 5 and 7 minutes). I sent this samples to Bioanalyzer before first strand cDNA synthesis and the profile is really weird. There's a peak around 200 bp (so the fragmentation seems to be good, especially 5 and 7 min: 2 was not enough) but the peak itself and the baseline are very noisy (distorted). I've read that can be a problem with the chip itself, but also because of not enough washing after the fragmentation. The thing is that the protocol I'm following doesn't not incluse a washing after this fragmentation. What do you think about this profile?. Any suggestion of what to do with these samples?. Thanks!

[nucacidhunter]

You would expect to see a smooth electropherogram if sample does not contain much salt. Samples containing first strand buffer (especially in sensitive Pico Chip) are expected to run as the profiles shown. Also, the actual sizes will be a bit larger than shown because salts accelerate nucleic acids migration in Chip.

There is no need to do a clean-up if the protocol does not call for it because the buffer composition would be part of the next step in protocol. RT reaction will be primed with random hexamers and because they will prime from different regions from RNA the final library inserts will be shorter than RNA fragment length. I think you can go ahead following the kit protocol.