Hi,
I am extracting RNA from bean shoots. Once I analyze it with the Bionalyzer, I observe an extra-peak in the precursor region. Someone would know which would be the reason? My RNA would be good to perform transcriptomic analyses? Thank you in advance.
Dear Laura
without seeing the trace is hard to say for sure
However, in general before the 2 principal ribosomal peaks you have smaller 5s rRNA & one of the internal size standards
Occasionally, you will also see a more prominent peak migrating in the same region: If this is so then it can be the GITC present in the RNA lysis buffer. If you see a prominent peak and a 260/230 ratio by Nano drop of < 1.0 that might be concerning as GITC is present in sufficient levels to inhibit down stream enzymes and prevent transcriptional analysis
In the past when I was processing a lot of RNA for Affymetrix RNA's I would see samples with this extra prominent peak and crucially a 260/230 ratio of < 1.0 implying GITC contamination. These samples hardly ever performed in down stream analysis. In this situation I would subject them to a 2nd round of RNA purification, whereupon the extra small peak disappeared; the 260/230 ratio by Nano drop was rendered > 1.0 and the samples started to perform in down stream analysis
Dear Laura,
We can not forget that the 2 principal rRNA peaks are not the rule for all organisms (e.g., trypasonomatids, insects under denaturing conditions).
Best,
Michele
Agreed Michelle. A few years back I extracted RNA routinely from Tryps and that was 3 peaks. However, the vast majority encountered in common research contexts are indeed 2
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