- You’ve done the hard work – you’ve got good quality RNA. You’ve seen ribosomal bands on a gel (or *cough* Fragment Analyzer). A good RQN/RIN (if that’s what you go by) and everything’s great.
- Now to get rid of the DNA
- Quick check (perform a PCR directly on appropriate amount of RNA) .. .and often there’s still some DNA.
- Another DNase I step . .another check and . .go to step 2
Yep, why are the ‘easiest’ steps sometimes the most time consuming?
So let’s look at the process – onen nearly always requiring DNase I enzyme. Yes, there are various kits that promise solutions that will selectively remove DNA but you’re probably going to find a DNase I digest is required at the end anyway. The solution-based digests usually seem to work more efficiently than the on-column digests (column fibres protecting DNA from digestion?).
I became interested in DNase I action due to a common tech question in a former life . . .the DNase I enzyme we sold had no buffer – it was just the enzyme. And thus a lot of questions came on ‘how do we use it’?
A number of interesting papers have been published over the years on the best buffers, temperatures . .and ways to kill DNase I. Some of them from my files include:
- BioTechniques 20:1012-1020 (June 1996) – compared inactivation temperatures for DNase I and showed 75-85°C was optimal. However no stop buffer was used in this work.
- BioTechniques 22:1128-1132 (June 1997) – using a reaction buffer containing Mn rather than Mg in order to get blunt cut, non-overlapping fragments . . .as well as boost the activity of DNase I.
- BioTechniques 29:38-42 (July 2000) – further work on DNase I inactivation by use of DTT and/or buffers of a non-neutral pH
- BioTechniques 29: 252-256 (August 2000) – preventing metal-induced hydrolysis of RNA by first using a stop buffer to chelate metal cofactor ions prior to the heat-kill step. Indicated that heat-killing without a stop buffer (even at the 75°C above) was detrimental to the RNA.
Many commercial systems (including the Quanta one) use this last method of stop buffer plus heat kill – allowing the DNase-treated RNA to be used directly in the cDNA synthesis step without any further purification. The Quanta system has been released in order to provide seemless matching of ion co-factors in the DNase I step with the concentrations in the cDNA supermix or the new XLT cDNA supermix.
So, given the required cofactors for DNase I activity (and subsequent inactivation), it can be worth using an appropriate buffer for RNA storage ie using low EDTA buffers to prevent chelation of ions required for DNase I activity.
If you have any modified protocols or procedures for DNase digestions then look forward to hearing about them in the comments below