Hi!
We've been extracting RNA from S. cerevisiae samples grown in synthetic media and in our RNA screentape data we see a large peak below 200 nt that keeps appearing. The RIN values and purity of the samples are all fine. The samples also pass the QC for illumina sequencing and subsequent library preparation. RNA was isolated using an acid phenol/chlorophorm/IAA protocol. At first i thought this was degradation, but it the rRNA peaks seem fine and it doesn't look like your typical degradation profile. Does anyone know what this peak represents?
Thank you!
Hi there,
These small size RNA are mainly tRNAs and probably 5S RNAs too. If you want to get rid of them you should use IEC (small RNA being eluted at lower ionic strength than rRNAs). Nucleobond RNA/DNA kit is what I'm using to separate differents RNAs.
Dear Denzel Ignacia
It is normal to obtain this sharp peak below 200 nt from yeast total RNA sample. This peak contains mainly small ribosomal RNAs like 5s rRNA (121 nt) and 5.8 rRNA (158 nt), tRNAs as these are abundant in total RNA sample.
The further sharp lower band around 15 bp/25 bp is not the part of your sample. It is called lower marker. This is added for sizing purpose in quality control. It is excluded in concentration measurement. It will not interfere with the library preparation and RNA sample sequencing.
Best wishes.
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