Hello everyone,
My name is Franz, I'm new to seqanswers.com, and also pretty new to NGS in general, so I hope my questions aren't too stupid.
Well, I want to do miRNA sequencing on Ion Torrent platform, using their Ion Total RNA seq kit v2.
I seem to have gotten it to a point where it seems to work fine, except this huge peak just below the miRNA inserts keeps popping up all the time. I know its not adaptor dimers, those should be smaller. In the case of the Ion Torrent kit, the adaptor dimers should be ~77nt, and the miRNAs ~97nt. I can get rid of the dimers using the included magnetic beads, and I get the miRNA peak, however, the additional peak I'm having is at ~87nt (insert-length ~10).
I do not have smallRNA chips for the bioanalyzer, so I can't say if it isn't simply actual RNA inserts (would degradation fragments actually look like this?), and it also might be the case that the input amount is not right. I am extracting the RNA from fresh-frozen human tissue using the Invitrogen PureLink miRNA kit. The input amount should be 1-100ng miRNA, with 5-25ng being ideal, I varied the input amount from 60ng smallRNA to 300ng, perhaps its not enough?
Also, I put some libraries on the bioanalyzer right after the final amplification step before size-selectioning, and it seems that the beads work for the dimers and some of the tRNA inserts above, but not for that 87nt peak, is it simply too close?
I have attached a PDF with bioanalyzer traces. I know the majority of people don't use Ion Torrent, but I believe the other manufacturer's kits work similarly. Does anyone here have any idea what this might be? A peak right between the adaptor dimers and the miRNA inserts?
regards,
Franz
My name is Franz, I'm new to seqanswers.com, and also pretty new to NGS in general, so I hope my questions aren't too stupid.
Well, I want to do miRNA sequencing on Ion Torrent platform, using their Ion Total RNA seq kit v2.
I seem to have gotten it to a point where it seems to work fine, except this huge peak just below the miRNA inserts keeps popping up all the time. I know its not adaptor dimers, those should be smaller. In the case of the Ion Torrent kit, the adaptor dimers should be ~77nt, and the miRNAs ~97nt. I can get rid of the dimers using the included magnetic beads, and I get the miRNA peak, however, the additional peak I'm having is at ~87nt (insert-length ~10).
I do not have smallRNA chips for the bioanalyzer, so I can't say if it isn't simply actual RNA inserts (would degradation fragments actually look like this?), and it also might be the case that the input amount is not right. I am extracting the RNA from fresh-frozen human tissue using the Invitrogen PureLink miRNA kit. The input amount should be 1-100ng miRNA, with 5-25ng being ideal, I varied the input amount from 60ng smallRNA to 300ng, perhaps its not enough?
Also, I put some libraries on the bioanalyzer right after the final amplification step before size-selectioning, and it seems that the beads work for the dimers and some of the tRNA inserts above, but not for that 87nt peak, is it simply too close?
I have attached a PDF with bioanalyzer traces. I know the majority of people don't use Ion Torrent, but I believe the other manufacturer's kits work similarly. Does anyone here have any idea what this might be? A peak right between the adaptor dimers and the miRNA inserts?
regards,
Franz
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