RNase H

Ribonuclease H (RNase H) is an endoribonuclease that selectively hydrolyzes phosophodiester bonds in the RNA strand of RNA-DNA hybrids. The enzyme is highly specific and does not digest single- or double-stranded DNA and does not digest unhybridized RNA. Divalent metal cations (typically (Mg²⁺) are required to drive catalysis.

QIAGEN RNase H is purified from a recombinant E. coli strain carrying the RNase H (rnh) gene from E. coli.

Supplied in: 
20 mM Tris-HCl, 100 mM KCl, 0.1 mM DTT, 10 mM MgCl₂, 0.1 mM EDTA, 50% glycerol (pH 7.9 at 25°C)

Supplied with:
10X RNase H Buffer (B9220): 500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl₂, 100 mM DTT (pH 8.3 at 25°C)

Use Thermostable RNase H (coming soon) to increase stringency, minimize nonspecific binding, and in applications where higher incubation temperatures are required.

RNase H is tested for purity, activity and the absence of contaminating endonucleases, exonucleases and other RNases.

• Storage temperature: –25°C to –15°C
• Molecular weight: 18.1 kDa

One unit is defined as the amount of enzyme that will hydrolyze 1 nmol of RNA from a 3H-labeled DNA-RNA hybrid molecule into acid-soluble material in 20 minutes at 37°C.

Ribonuclease H is an endoribonuclease that specifically hydrolyzes the RNA strand in RNA-DNA heteroduplexes. Hydrolysis of the RNA phosphodiester bond results in the formation of 3′ hydroxyl and 5′ phosphate groups. The enzyme requires divalent metal cations, typically Mg²⁺ to drive catalysis. E. coli RNase H belongs to the RNase H1 class of enzymes present in prokaryotes, eukaryotes and the domains of reverse transcriptases. 

Ribonuclease H enzymes are required for DNA integrity and are involved in fundamental cellular processes including removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. The strict selectivity of RNase H makes it indispensable to molecular biology workflows where cleavage of RNA in RNA-DNA hybrids is required. The enzyme is widely used in RT-PCR and cDNA synthesis to remove messenger RNA (mRNA) after first-strand cDNA synthesis.

Usage Instructions

1. Set up the following reaction mixture in a total volume of 100 µL: 

   Components	Final Concentration	Volume
   Nuclease-free water	N/A	X µL
   10X RNase H Buffer (B9220)	1X	10 µL
   RNA: DNA duplex	2 µg	X µL
   RNase H (Y9220L)	5 U	1 µL
   	Total Volume =	100 µL
2. Incubate the reaction at 37°C for 20 minutes. 

RNase H is an endoribonuclease specific for degradation of RNA in RNA-DNA heteroduplexes without digestion of the DNA strand. This strict selectivity makes RNase H an indispensable addition to molecular biology workflows for cDNA synthesis, RNA structural analyses and other applications where cleavage of RNA in RNA-DNA hybrids is required.

• cDNA synthesis: RNase H degrades mRNA following first-strand cDNA synthesis to enable synthesis of the second DNA strand
• RT-PCR: RNase H removes RNA templates after reverse transcription to improve downstream DNA amplification
• RT-qPCR: RNase H enhances specificity and performance through controlled degradation of RNA in RNA-DNA hybrids
• Post–reverse transcription cleanup: Selective action of RNase H prepares cDNA for downstream enzymatic reactions
• Site-specific cleavage of RNA within RNA-DNA hybrids: RNase H facilitates mapping of RNA structure or probing specific RNA regions