RNase A, Bovine pancreas

Application Method of RNase A, Bovine Pancreas in Ribonuclease Protection Assay (RPA)^[1]

This experimental protocol is compiled based on research content and is for reference only; please adjust according to actual needs.

(1) Reagent preparation: Dissolve RNase A, Bovine pancreas in an appropriate buffer (e.g., 10mM Tris-HCl, pH7.5, 300mM NaCl, 5mM EDTA) to prepare a stock solution (e.g., 10mg/mL); mix with RNase T1 to prepare the RNase digestion mixture (final concentrations typically RNase A, Bovine pancreas 40μg/mL, RNase T1 2μg/mL); prepare radiolabeled (e.g., ³²P-UTP) antisense RNA probes (length 150-500nt), purify and set aside; synthesize unlabeled sense RNA (cRNA) at known concentrations as absolute quantification standards.

(2) Probe and sample preparation: Clone the cDNA fragment corresponding to the target mRNA into a plasmid containing a phage RNA polymerase promoter (SP6, T3 or T7); linearize the plasmid with restriction enzyme digestion, then perform in vitro transcription to obtain antisense RNA probes (specific activity approximately 10⁹cpm/μg); total RNA samples (typically 1-10μg) are used directly for liquid hybridization without electrophoretic separation.

(3) Hybridization: Mix an appropriate amount of radiolabeled probe (approximately 5×10⁵cpm) with the RNA sample (or serially diluted cRNA standards) in hybridization buffer; incubate at a suitable temperature (usually 42-45°C) overnight (12-16h) to allow sufficient formation of RNA-RNA hybrids between the probe and target mRNA.

(4) RNase digestion: After hybridization, add the RNase digestion mixture (containing RNase A, Bovine pancreas and RNase T1); incubate at 30-37°C for 30-60min to specifically degrade unhybridized single-stranded RNA probe and sample RNA; then terminate digestion by adding proteinase K and SDS; extract with phenol/chloroform and precipitate with ethanol to recover RNase-resistant hybrids.

(5) Product analysis: Dissolve the precipitated hybrids in loading buffer, separate by denaturing polyacrylamide gel electrophoresis (PAGE), dry the gel, and detect protected fragment signals by autoradiography or phosphor imaging; alternatively, directly precipitate RNase-digested hybrids onto filters for scintillation counting quantification. Compare with the cRNA standard curve to determine the absolute content of target mRNA in the sample.

Precautions:

(1) RPA sensitivity is at least 10-fold higher than Northern blot, allowing detection of low-abundance mRNA (as low as 5pg).

(2) The optimal probe length is 150-500nt; too short results in weak signals, while too long tends to produce smeared bands due to radioactive decay-induced fragmentation.

(3) For your safety and health, wear laboratory coat and disposable gloves during operation; radioactive isotopes must be handled in accordance with regulatory protection measures.

References:
[1] Reue K. mRNA quantitation techniques: considerations for experimental design and application. J Nutr. 1998 Nov;128(11):2038-44.