Choosing an rnase inhibitor and using dnase i properly: which inhibitor class survives your buffer, why the enzyme you add to protect RNA can also destroy it, and the order these two steps belong in

Getting both enzymes right

1. Know which inhibitor class you are buying. Protein ribonuclease inhibitors bind the enzyme tightly and are effective and fragile: they lose activity below about pH seven, above roughly fifty degrees, and in the presence of reducing-agent-free buffers, because they depend on free thiols. Chemical inhibitors are more robust and less specific.
2. Keep the reducing agent in the buffer. A protein inhibitor oxidises and dissociates without a reducing agent present, at which point it releases the ribonuclease it was holding. A buffer prepared without one is worse than no inhibitor at all, because the protection was assumed.
3. Do not heat it and expect it to work. Reverse transcription at the higher temperatures some enzymes allow is outside the protein inhibitor's working range. Where the protocol runs hot, the inhibitor is protecting the sample before the reaction, not during it.
4. Use dnase i to remove genomic DNA, deliberately. Residual genomic DNA is the usual cause of a signal in a no-reverse-transcription control. A DNase step during or after purification removes it, and a no-RT control on every experiment is what proves the step worked.
5. Remove the nuclease without boiling the RNA. Heating DNase I in the presence of magnesium degrades RNA. Inactivate by chelating the magnesium, by a column or bead cleanup, or with a removal reagent, and treat a 95 degree kill step as the thing that ruined the sample rather than as a protocol.

The bench practice that matters more than either reagent

Ribonucleases are on skin, on dust and on every surface, and they are extraordinarily stable. A dedicated RNA area, dedicated pipettes, filter tips, gloves changed often and certified nuclease-free plasticware prevent more degradation than any additive.

Decontaminating surfaces with a purpose-made reagent, and not reusing glassware for RNA work without baking it, are the two habits that separate laboratories where RNA work is routine from laboratories where it is a struggle.

Measuring integrity honestly

A capillary electrophoresis integrity number is the standard and a gel is a coarse substitute. Measure integrity on the sample that goes into the experiment, not on an aliquot taken three steps earlier.

Record the integrity number with the result. Where a downstream experiment behaves oddly, the first useful question is whether the input was intact, and the answer should not require re-running anything.

Buying nuclease-free

Certified nuclease-free water, tips and tubes cost a little more and remove a whole class of intermittent failure. The certification is per lot, and keeping the lot number with the experiment makes a contamination episode traceable rather than mysterious.

Aliquot water and buffers into single-use volumes. A shared bottle dipped into repeatedly is the commonest point of contamination in an otherwise careful laboratory.

Common questions

Which rnase inhibitor should I use?

A protein inhibitor for standard reactions near neutral pH and moderate temperature, with a reducing agent in the buffer. A chemical inhibitor where conditions are harsher or where you cannot guarantee the buffer, accepting that it is less specific.

Why did my RNA degrade despite adding an inhibitor?

Most often the buffer had no reducing agent, the pH was below the inhibitor's working range, or the reaction ran hot. All three release the inhibitor from the ribonuclease it was holding, which is worse than never adding it.

Do I always need dnase i?

Whenever the assay cannot distinguish RNA from DNA, which includes most quantitative PCR against genes with no intron between the primers. Run a no-reverse-transcription control and let that decide rather than assuming.

How should DNase be inactivated before reverse transcription?

By removing the magnesium or the enzyme, not by heating. Heat plus magnesium fragments RNA, and this single step accounts for a great deal of unexplained loss of integrity between extraction and reverse transcription.

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