How to quantify dsRNA in IVT samples using MDA5-ATPase assay

Step-by-step instructions for quantification of dsRNA in IVT samples using MDA5-ATPase assay

Materials and Instruments

• Instruments

• Reagents

• Malachite green oxalate (MG) buffer

To make a solution of 13mM Malachite green oxalate, 3.6M sulfuric acid is used as a solvent to dissolve the Malachite green oxalate. Store in a dark place.

• Working buffer

To prepare a working buffer, mix MG buffer, 7.5% ammonium molybdate tetrahydrate, and 11% Tween20 are mixed in a volume ratio of [1: 0.25: 0.02]. The resulting master mix is then diluted with 4 volumes of ddH2O. It is important to note that the working buffer should be prepared freshly, as it may not be stable over time.

• Reaction buffer:

20 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT.

Experimental Procedure

Quantification of purified RNA

1. Use the Nanodrop spectrophotometer (Thermo-Scientific, 840-317400) to measure the absorbance of purified RNA at 260nm.
2. Record the absorbance value.
3. Calculate the RNA concentration using the Nanodrop software.

Binding of IVT samples with MDA5-ΔN

1. Add the calculated amount of purified IVT samples to the reaction system containing MDA5-ΔN.
2. Incubate the reaction mixture at 37°C for 15 minutes.
3. Stop the reaction by adding 5μL of 0.5M EDTA.

Spectrophotometric analysis of RNA-MDA5-ΔN interaction

1. Mix 10μL of the stopped reaction mixture with 90μL of working buffer at room temperature for 30 minutes.
2. Measure the OD620nm absorbance of the mixture using the LUX machine (Thermo Scientific, VLB000D0).
3. Record the absorbance value.