View Single Post
Old 10-21-2016, 12:52 PM   #4
JDSwenson
Junior Member
 
Location: Bay Area, CA

Join Date: Apr 2016
Posts: 8
Default

Thank-you for the replies!

I've called tech support and I ran a time series, fragmenting at 5, 7, 10, and 12 minutes. Then I did a cleanup with 2.2x AmPure beads following the RNAClean XP protocol. Attached are my results from the fragmentation time series -- three of the four fragmentations resulted in two distinct peaks, but the fourth looks totally different. Has anybody seen these sorts of sharp peaks after a fragmentation before?

The time for which the mRNA was fragmented at 94 degrees C is in the sample name (e.g. JS16_5 is 5 minutes, JS16_12 is 12 minutes). Everything else was kept the same, including the source of the input RNA.

Parting thoughts ...
I did my original RNA extraction with Trizol followed by a column-based purification and I've read that Trizol often preserves small RNAs, which would explain the ~200 bp peak. My mRNA isolation used oligo d(T) beads, so there shouldn't be any 5s RNAs in the sample (right?) ... but that still doesn't explain why three of the four fragmentations produced peaks instead of curves nor why the length of the fragments seems to have no correlation to fragmentation time ... Any ideas?

Any input whatsoever would be appreciated. I'm pretty baffled.

Thanks a lot
Attached Files
File Type: pdf mRNA_test_Sample3_diff_frag_times.pdf (1.05 MB, 11 views)
JDSwenson is offline   Reply With Quote