Estimation of RNA | Biotechnology

In this article we will discuss about the principle, requirements and procedure for estimation of RNA.

Principle:

Ribose (a pentose) in presence of hot acid is converted to furfural which then reacts with orcinol to yield a green colour, read at 660 nm.

Requirements:

1. Orcinol:

Prepare 10% (W/V) FeCl₃. 6H₂0 solution. To 0.5 ml of this FeCl₃ solu­tion add 1 g of orcinol with concentrated HCl, make up the volume to 100 ml.

2. Standard RNA:

Dissolve 100 g in dis­tilled water with addition of a few drops of 0.1 (N) NaOH and make up the volume to 100 ml.

3. Sample RNA (isolated).

4. 0.1(N) HCl.

5. Test tubes and stands.

6. Pipettes (graduated).

7. Water bath 100°C.

8. Spectronic 20.

9. Glass marking pencil.

10. Distilled water.

Procedure:

1. Take 0.5,1.0,1.5,2.0 and 2.5 ml of stand­ard RNA. 0.5 ml of sample RNA and 1.0 ml of 0.1 (N) HCl (blank) and label.

2. Add 1.0 ml of 0.1 (N) HCl to all tubes except blank.

3. Add distilled water to all tubes to make 3.5 ml.

4. Add 3 ml orcinol to all tubes, shake well and heat in a boiling water bath for 20 minutes.

5. Cool the tubes and read absorbance at 660 nm.

6. Plot a graph with standard concentra­tion against absorbance and find out the amount of RNA in sample.