Ribonucleic Acid - Assay

A320 is a check for extraneous background.

Calculation of % Native RNA:

RNase Blank Assay: (Can be set up with RNase assay) Set up a blank for sample RNA and a blank for test RNA. Pipet one ml of 0.10M sodium acetate buffer pH 5.0 into centrifuge tubes. Equilibrate to 37°C. Add one ml of RNA Solution (2.5 mg/ml). Let set one hour at 37°C. Then add one ml of uranyl acetate-perchloric acid solution. Transfer to an ice bath and cool for 5 minutes. Clarify by centrifugation and dilute 0.1ml of clear supernatatnt to 3.0ml with reagent grade water. Read A260 vs blank. Blank should not increase more than 50% in one hour at 37°C.

RNase Assay: Set up one set of tubes for Sample and one for Control. Include a Blank with the Sample set of tubes, and a Blank with the Control set of tubes. Pipette one ml of respective enzyme dilution into centrifuge tubes; pipette one ml of 0.10M sodium acetate buffer pH 5.0 into Blank tubes. Incubate all tubes at 37°C for 58 minutes. At timed intervals, add one ml of RNA Solution (2.5 mg/ml) to all tubes (Samples, Controls, and Blanks). Incubate each tube exactly 4 minutes and stop reaction by the addition of one ml of uranyl acetate-perchloric acid solution. Transfer to an ice bath and cool for 5 minutes. Clarify by centrifugation and dilute 0.1 ml of clear supernatant to 3.0 ml with reagent grade water. Read A260 versus blank.