Estimation of RNA

Estimation of RNA

Aim

To estimate the amount of RNA in the given sample

Principle

Acid hydrolysis of RNA releases ribose which in the presence of strong acid undergo degradation to yield furfural.  Orcinol in the presence of ferric chloride reacts with furfural producing a green coloured compound with absorption maxima at 665 nm.  This is a genera reaction for pentoses.

Materials required

1. 6% orcinol – 6 gm of orcinol in 100 ml of 95% ethanol.  Keep the solution in brown bottle at 4^oC

2. 2 ml of 10% ferric chloride added to 400 ml concentrated HCl in brown bottle

3. RNA standard solution – 100 µg RNA/ml distilled water

Procedure

1. 0.2, 0.4, 0.6, 0.8 and 1 ml of Standard RNA solution was pipetted out into a series of test tubes.

2. 1 ml of the unknown RNA solution was pipetted out into another test tube.

3. The volume was made up to 1 ml in all test tubes using distilled water. A tube with 1 ml distilled water served as the blank.

4. 1 ml of ferric chloride solution and 0.2 ml of orcinol reagent was added to each tube

5. The mixture was heated in a boiling water bath for 15 minutes and cooled at room temperature.

6. The green colour developed was measured at 665 nm against blank.

7. A standard graph was plotted and amount of RNA in the given sample was calculated.

Observation

Sl No.	Volume of RNA solution (ml)	Volume of distilled water (ml)	Volume of ferric chloride solution (ml)	Volume of orcinol reagent (ml)	Concentration of RNA (µg)	Optical density at 665 nm
1	0.0	1.0	1.0	0.2	0	……..
2	0.2	0.8	1.0	0.2	20	……
3	0.4	0.6	1.0	0.2	40	…….
4	0.6	0.4	1.0	0.2	60	…….
5	0.8	0.2	1.0	0.2	80	……
6	1.0	0.0	1.0	0.2	100	…….
7	1.0	0.0	1.0	0.2	……	…...

Calculation

From graph, concentration of the given sample is

By calculation

OD of the test  = Concentration of test

OD of standard Concentration of standard

Concentration of the given sample is =

Result

The amount of RNA in the given sample

From graph =

From calculation =