I'm working with very small samples and I'm wondering if I should include the on column DNAse step or leave it out. I know the columns should do a good job even without this, and I've found a few times that it greatly reduces my yield (more than half sometimes).
I'm planning to use the RNA for bulk RNAseq, so I have to hit a threshold for RNA concentration. Thanks!
Hi Helen
Its right that the DNAse astep is optional. However, if not using it still gives you a good RIN number for RNAseq, its better to skip this step.
It would also be better to speak to the RNAseq service provider to understand if they have special recommendations regarding DNA free samples.
Hope this helps.
Best
Aparajita
Could it not be reducing your yield because...quite a lot of your yield is DNA?
I mean, if you're dealing with small samples, you might not have much RNA anyway, and any DNA contamination will add to the measured "apparent" RNA concentration. Digesting away this DNA will lower the measured nucleotide content without necessarily changing the total amount of RNA.
Honestly, RNAseq is pretty expensive, and any carryover DNA will reduce the quality of your data, especially if your RNA yields are low.
Aparajita Lahree John Hildyard Thanks so much for your responses.
Still getting decent RIN numbers for some of my samples, degradation seems to be more of an issue than DNA contamination. We'd been skipping the beta mercaptoethanol step, so adding this in should hopefully help a bit with that.
I'm going to try a few bigger samples with and without the DNAse step and see how we get on in terms of yield. We have access to a tapestation now so can get a pretty good idea.
Thanks again!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA