Hello all,
I was wondering if there is a negative effect of RNase inhibitors (like RNase Out, RNasin, etc) on low input RNA samples at higher concentrations? I find that many single-cell RNAseq methods are using concentrations as low as 0.01U/uL where standard RNA-seq protocols are using 1U/uL. Or instead is this simply to avoid reagent waste, i.e. less RNase inhibitor needed for less RNA?
Thanks!
Morgan
The way it works, RNasin tightly binds to some ribonucleases and blocks their active sites. Commercial preps represent a recombinant RNasin, which might contain traces of RNasin-RNase complexes originating from interactions between the overexpressed RNasin and RNases of the host. If your RNasin-protected RNA samples do not undergo a heat treatment, such a contamination may not reveal itself. However, if you have to heat them above 50-60 oC, RNasin will be denatured while RNases may still retain their activity with obvious consequences for RNA integrity. Thus, high RNasin loads may paradoxically be more dangerous for your RNA than no RNasin at all (but only for scenarios involving RNasin denaturation). Therefore, people try to keep its concentration at minimum.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA