Ambion RNase Inhibitor, a recombinant human protein produced in E. coli, is a potent inhibitor of neutral pancreatic RNase A type enzymes. The mode of inhibition is noncompetitive; the inhibitor tightly binds RNases in a 1:1 ratio. The enzyme has been shown to inactivate RNases present in many tissues and cell types. Addition of the ribonuclease inhibitor (RI) has been shown to be useful whenever the integrity of RNA must be maintained such as in the preparation of cDNA by reverse transcription, in vitro RNA transcription, and in in vitro protein synthesis. RI does not inhibit RNase I, T1, T2, H, U1, U2, or CL3. RI requires a minimum of 1mM DTT to maintain activity, and has an activity range between pH 5.0 to 8.0 with maximal activity between pH 7.0 and 8.0. Since the mode of inhibition is the formation of a 1:1 complex with RNases, care must be taken to avoid denaturation or oxidation of the ribonuclease inhibitor (e.g., by addition of SDS, urea, etc.). RI should be added to transcription, translation, and cDNA synthesis reactions to give a final concentration of 1U/μL. RNase Inhibitor is rigorously tested for contaminating RNase, exonuclease, endonuclease, and protease activity. Unit Definition: One unit is the amount of protein required to inhibit the activity of 5ng of RNase A by 50%. Unit assay conditions: 100mM Tris-acetate (pH 6.5), 1mM EDTA, 1mM cyclic 2 ft.,3 ft.-CMP, and RNase inhibitor. Addition of RNase A initiates the reaction. RNase Inhibitor activity is measured by the inhibition of hydrolysis of cyclic 2 ft., 3 ft.-CMP by RNase A.
Gene Expression Analysis and Genotyping, In Vitro Transcription, PCR and Real-Time PCR, Real Time PCR (qPCR), Reverse Transcription