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Abstract

Methods for preparing RNA from ribonuclease-rich sources while avoiding RNA degradation are described. The lysis protocol for ribonuclease-containing samples is performed at high pH to accelerate cell lysis and with a reducing agent that inactivates ribonucleases (RNases) by reducing disulfide bonds essential for RNase activity. Samples are briefly incubated for up to five minutes at high pH followed by addition of a reagent to lower the pH to a level at which the RNA is stable. This method of RNA extraction has many advantages over existing methods of RNA preparation, including that cell lysis is efficient, RNases are rapidly inactivated, and sample incubation times are short (less than 5 minutes), which protects RNA from degradation. The lysing procedure is performed entirely in aqueous solution with no heating, precipitations, or buffer exchanges required. Thus, a quick, simple procedure for extracting RNA is provided, which can easily be automated.

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