RNase Inhibitor Hu

• Completely inhibits RNase A, RNase B, and RNase C activity
• Does not influence any polymerase or reverse transcriptase activity
• Retains full stability at 37°C for at least 4 weeks with no loss of activity
• Active in diverse reaction conditions and in various buffers
• Free of DNase and RNase activity

RNase Inhibitor Hu is a thermally resistant, 50 kDa recombinant human placental protein expressed in Escherichia coli that inhibits RNase activity of common eukaryotic enzymes such as RNase A, RNase B, RNase C.

It is supplied with 20 mM Hepes-KOH (pH 7.6), 50 mM KCl, 8 mM reducing agent, 50% (v/v) glycerol. It is stable up to 58°C and at a minimum 0.5–1 mM DTT concentration ranges.

One unit is defined as the amount of enzyme required to inhibit the activity of 5 ng RNase A by 50%.

RNase Inhibitor Hu inhibits RNases by non-covalent binding in a 1:1 ratio with high-affinity protein-protein interaction, forming one of the tightest known biomolecular complexes. This activity effectively helps in the maintenance of RNA integrity and in obtaining the appropriate quantity and quality of RNA.

The enzyme is used in applications where the presence of RNases causes a substantial hazard in receiving good quality, trustworthy, and reproducible data, e.g., in RNA isolation, cDNA synthesis, RT-PCR, in vitro transcription and translation, or RNase-free monoclonal antibody preparation [1-6]. It is compatible with numerous reagents, including DNA polymerases and AMV or M-MuLV Reverse Transcriptase, and its addition into the reaction does not influence the efficiency of other components.

Quality Control

Protein purity is determined using assay by SDS-PAGE electrophoresis resulting in >90% purity.

RNase contamination is evaluated by assessing RNase activity through incubation of 1 µg of RNA with a minimum of 200 U of the RNase Inhibitor for 1 hour at 37°C. Results are visualized on an ethidium bromide-stained agarose gel.

Latent RNase contamination is evaluated by assessing latent RNase activity through incubation of 1 µg of RNA with a minimum of 200 U of heat-inactivated RNase Inhibitor for 1 hour at 37°C. Results are visualized on an ethidium bromide-stained agarose gel.

Endonuclease activity is determined by incubating 1 µg of supercoiled plasmid DNA with a minimum of 200 U of the RNase Inhibitor for 2 hours at 37°C. Results are visualized on an ethidium bromide-stained agarose gel.
Exonuclease activity is determined by incubating 1 µg of digested plasmid DNA with a minimum of 200 U of the RNase Inhibitor Lyo-ready for 2 hours at 37°C. Results are visualized on an ethidium bromide-stained agarose gel.

Usage

• The optimal final concentration of the RNase Inhibitor Hu in a reaction depends on the level of RNase contamination, the incubation time and the compounds present in the reaction mixture. It falls within a range of 1–2 U/μL.
• For a standard reverse transcription reaction, use 1 μL (40 U) of the RNase Inhibitor Hu for a final sample volume of 20 μL.
• A final DTT concentration of 0.5–1 mM is essential for optimal activity.
• During the reaction assembly, the enzyme should be added before other components that are possible sources of RNase contamination.
• Using RNase Inhibitor Hu does not exclude RNase H treatment after amplification of the first strand cDNA.

This is used for applications to overcome the challenges in inhibiting the presence of ubiquitous RNases, commonly found in skin, dust, reagents, and biological samples, such as:

• RNA-related molecular diagnostics
• RNA isolation and purification
• cDNA synthesis, RT-PCR, RT-qPCR
• in vitro transcription and translation
• RNase-free monoclonal antibody preparation