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.

rnase free water, and the rest of an RNase-free bench

RNase free water is the cheapest part of keeping RNA intact and the part most often blamed. Water sold for the purpose is tested for nuclease activity rather than treated with an agent, since a residual inhibitor would follow the water into the reaction. The rest of the bench is where samples are actually lost: certified tips and tubes, gloves changed often, surfaces and pipette barrels wiped with a decontamination solution, reagents aliquoted so a shared stock cannot be spoiled once, and a separate area away from plasmid and protein work. An inhibitor in the mix is a safeguard, not a substitute for any of it.

rnase free dnase, and the dnase rnase pair on a bench

Removing DNA from an RNA preparation means adding a nuclease, which is exactly the thing an RNA bench spends its effort excluding, so the enzyme has to be certified free of ribonuclease activity and then removed or inactivated completely. Column-based digestion or a heat-labile enzyme avoids the phenol step that a carried-over protease treatment would need. The general rule on the bench is that the two enzyme families are kept physically apart, with their own tips, tubes and pipettes, because one contaminated stock costs a month of samples.

rnase a powder and the contamination it becomes

rnase a powder is bought to digest RNA deliberately, and it is also the most persistent contaminant in a laboratory that uses it, because the enzyme is small, stable and survives autoclaving. Weighing it in a room where RNA work happens is how an RNA preparation stops working. A dedicated area, dedicated tools and a stock solution rather than powder are the controls.

A t7 endonuclease and the mismatches it cuts

T7 endonuclease I cleaves at mismatches and heteroduplexes, which is why it is used to estimate editing efficiency after reannealing amplified DNA, and why the assay is semi quantitative: it misses small indels and is confounded by polymorphisms. Sequencing has largely replaced it for a final number, and the enzyme remains useful as a quick screen.

lysc and a protease for defined cleavage

Lys-C cleaves after lysine and tolerates urea, which is why it is used before trypsin in proteomics digests and where a protein resists denaturation, and its specificity is what makes a peptide map interpretable. Incubation time, temperature and the enzyme to substrate ratio are what decide missed cleavages, and those numbers belong in a method rather than the enzyme's name.

marfey's reagent and the chirality it reveals

Marfey's reagent derivatises amino acids so that the two enantiomers separate on an ordinary reversed phase column, which is how a peptide's D and L residues are confirmed without a chiral column. That matters wherever a D residue is part of the activity. The reaction and the derivatives' stability decide the method, and standards of both forms are run alongside.

An atp solution and how it is kept

An atp solution hydrolyses on freeze thaw and at neutral pH over time, so the practical choices are small single use aliquots, a stated pH on preparation and a concentration confirmed by absorbance rather than assumed from the weight. Magnesium is required by most enzymes that use it, so the buffer's magnesium is part of the reagent rather than a detail.

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.

Browse by service class

• Compare Gene synthesis on advertised prices