1. I want to degrade all RNA in an sample, without damaging the DNA in the same sample. What buffer (pH), temperature and time should I use?
2. I want to preserve the 3' cyclic phosphate that is generated during the transesterification (often called hydrolysis) of the RNA. The cyclic phosphate is unstable in acid, but how stable is it in an alkaline buffer?
RNA is very easy to degrade. Just add DNase-free RNase from Roche (cat#11119915001) or from Life Tech (cat#EN0531) to your sample. Room temperature or 370C for 15-30 min. When Life Tech tests their enzyme, they used pH5 and incubated at 370C. Hope this helps.
Thanks but I want to degrade the RNA without RNAses, by incubating in an alkaline buffer. It's pretty standard, but I'm just wandering if anyone has any specific recomondations with respect to pH, temperature and time.
I did once at 90C for half hour in regular EB buffer, the RNA was only partially degraded, i believe increasing pH should help, but never tried it. Would like to know the best pH condition as well.
I definately helps increasing the pH. Also the concentration of Mg2+ has an effect. Life Tech / Ambion suggests this buffer: 1X Alkaline Hydrolysis Buffer (e.g., 50 mM Sodium Carbonate [NaHCO3/Na2Co3] pH 9.2, 1 mM EDTA; or the Alkaline Hydrolysis Buffer supplied with Ambion's RNA Grade Ribonucleases). My concern is that if you increase the pH to say 12, you'll also degrade the DNA in the sample........I really hope someone has the golden protocol ;-)
To hydrolyze RNA without damaging DNA, treat samples with 0.3 N KOH for 16 hr. at 37°C (Mathews, 1968).
Mathews CK (1968) Biochemistry of Deoxyribonucleic Acid-defective Amber Mutants of Bacteriophage T4: I. RIBONUCLEIC ACID METABOLISM J. Biol. Chem. 243(21) 5610-5615.
This paper studied the pH, Temperature, and Mg, K ion concentration effects to the RNA degragation.
J. Am. Chem. Soc. 1999, 121, 5364-5372
By the way, did you test the total RNA degragation with response to pH, Temperature and Time? If so, please recommend your related publications. Thank you.
I needed to hydrolyze RNA and preserve ssDNA.
For me nicely worked:
RNA-DNA hybrid in volume 60 ul
Hydrolyze RNA by the addition 12 ul of 0.5 M EDTA (pH 8.0) + 12 ul NaOH (1 M NaOH) followed by incubation a 95 °C for 10 min...
stop the reaction by addition 24 ul 0.5M HCl (it will neutralize solution to pH 7-8)
Notes:
EDTA (pH) has to be added first to chelate Mg2+, otherwise Mg2+ ions will precipitate in alkalic conditions and entrap DNA in precipitate.
pH above 11 should not hydrolyze DNA, only denaturate
0.5 M EDTA (pH 8.0) + 12 ul NaOH (1 M NaOH) will have pH above 13
I have compared the efficiency of RNA hydrolysis and ssDNA preservation with RNAse treatment and run ssDNA on alkalin Agarose gel (denaturing conditions) electrophoresis to check ssDNA quality. I have observed similar results.
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