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Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing the SP6 promoter.
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A method to detect and characterize point mutations in transcribed genes.

Ribonuclease A （RNase A） is an endoribonuclease, that specifically cleaves single-stranded RNA 3' to pyrimidine residues （cytosine, uracil）. Thereby, it generates pyrimidine-3'-phosphate or oligonucleotides with terminal pyrimidine-3'-phosphates. The pH-optimum is in the range of 7.0 - 7.5. RNase A is used for the purification of RNA-free DNA, for the removal of non-hybridized regions of RNA : DNA-hybrides or as a molecular weight marker. The enzyme is inhibited by diethyl pyrocarbonate （DEPC）, guanidinium salts （4 M GuaSCN）, β-mercaptoethanol, heavy metals, vanadyl-ribonucleoside-complexes, RNase-inhibitor from human placenta and competitively by DNA, respectively. Regarding the latter, the effect of denatured DNA is higher than by native nucleic acids. Nevertheless, RNase A is very active under very different conditions and difficult to inactivate. At low salt-concentrations （up to 100 mM NaCl）, RNase A cleaves single- and double-stranded RNA and RNA in RNA : DNA- hybrides. Under high salt concentrations （>300 mM NaCl） single-stranded RNA is cleaved only. To remove the enzyme from samples, it has to be digested by proteinase K （frequently, SDS at a final concentration of 0.6 % is added） and several phenol extractions are required. （Applications: Enzymatic manipulation of DNA and RNA: ref. 1 Suppl. 8 p. 3.13.1; minipreps of plasmid-DNA: ref. 1 Suppl. 24 p. 1.6.6; inSitu-hybridisation of cellular RNA: ref. 1 Suppl. 7 p. 14.3.8; removal of RNA from plasmid preparations: ref. 2 p. 1.51）
Stock solutions are prepared at concentrations from 1 - 10 mg/ml in 10 mM Tris · HCl, pH 7.5; 15 mM NaCl or in 10 mM Tris · HCl, pH 7.5; 1 mM EDTA, pH 8.0 （TE buffer）. The recommended working concentration is 10 μg/ml （removal of RNA from plasmid preparations; 1 hr, RT） or 100 ng/ml （preparation of "blunt ends" of double-stranded cDNA）.
Unit-definition: One unit of activity is defined as that amount of enzyme which causes the hydrolysis of RNA to yield a velocity constant, k = 1, at 25°C and pH 5.0 （Kunitz-Unit）.
Stability: RNase A aggregates during lyophilizing and storage. It has a high affinity to glas surfaces, which has to be taken into consideration. At neutral pH （e. g. in PBS pH 7.4） and high concentrations （> 10 mg/ml） the enzyme will precipitate. At +4°C （lyophilized） it is stable for several years （dry storage）, in solution （-20°C） several years or （+4°C） several weeks.