Extraction and Estimation of Ribonucleic Acid (RNA)

In this article we will discuss about the Extraction and Estimation of Ribonucleic Acid (RNA).

Principle:

The ribonucleoprotein complex is dissociated by SDS into RNA and protein, deproteinized by phenol and the free RNA left in aqueous solution is precipitated in the cold alter adding alcohol.

Materials:

a. Centrifuge (bench top)

b Magnetic stirrer

c. Cold room (4°C)

d. Phenol (freshly redistilled)

e. Extraction buffer (pH 9 0)

i. Tris-HCl (0-1 M) 1-21 g

ii. NaCl (0-075 M) 0-44 g

iii. EDTA Na₂ (0-005 M) 0-19 g

iv. Water 100 ml

f. Ethanol

g. SDS 10% (w/v in water)

h. Ether

i. Acid reagent

j. Standard RNA and extracted RNA sample

k. Spectrophotometer

All glassware should be baked at 120°C over­night. Contamination with any nuclease should be strictly avoided.

(A) RNA extraction procedure:

All operations should be done at 0-4°C.

1. Freeze 0-5-5 g of the material in a mortar and pestle with liquid N₂, grind to a fine powder, then to a paste and extract in 10 volumes of extraction buffer.

2. Centrifuge the homogenate at 2,000 × g for 3 min.

3. Transfer the supernatant to a volumetric flask and stir with 0-1 vol. of 10% SDS for 2-3 min.

4. Add an equal volume of buffered phenol (freshly redistilled phenol saturated overnight with 100 mM Tris-HCl pH 8-5).

5. Partition the content by centrifuging at 2,000 × g for 5 min and collect the upper aqueous phase into a separate flask.

6. Shake the lower and interphase again with an equal volume of extraction buffer for 5 min and centrifuge.

7. Combine the aqueous phase with the first one (step 5) and stir with an equal volume of buffered-phenol for 5 min.

8. Repeat the extraction and centrifugation steps at least five times or until the interphase shows no proteins.

9. Finally, collect the upper aqueous phase containing RNA, dissolve in it about 250 mg NaCl, add two volumes of cold ethanol (96%) and leave the flask overnight at -20°C for RNA precipitation.

10. Collect RNA by centrifugation at 2,000 x g for 10 min. Wash the pellet (RNA) with 70% ethanol, ethanol, ethanol : ether (1:1 v/v) and finally with ether. Dry the pellet gently ‘in vacuo’ for a few minutes.

11. Dissolve the RNA completely in elution buffer for further analysis by vortexing.

12. Dilute 20 µl aliquot to 2 ml with buffer and read the absorbance using 1 cm light path cuvette at 260 nm in a spectrophotometer. One A₂₆₀ unit is assumed equivalent to 40 µg RNA/ml. Otherwise, the RNA content is estimated colorimetrically.

(B) Procedure for measurement of RNA:

1. Prepare a standard RNA (50 µg RNA/ml) solution in ice-chilled 10 mM Tris acetate, 1 mM EDTA buffer (pH 7-2) or any other mitable buffer by dissolving RNA completely.

2. Dissolve the isolated RNA in the above buffer solution to an approx. concentration 50 µg/ml.

3. Prepare a series of tubes containing 0-5 ml, 1 ml, 1-5 ml and 3 ml of isolated RNA, 0-5 ml/1 ml, 1 -5 ml and 3 ml of 50 µg standards RNA/ml.

4. Make up each tube to 30 ml with water. In addition set a blank containing 3 ml of water.

5. Add 6 ml of orcinol acid reagent to each tube.

6. Add 0-4 ml of 6 0% alcoholic orcinol to each tube. Shake the tubes to mix the contents, and then heat all tubes in a boiling water bath for 20 min.

7. Cool the tubes, and read the absorbance at 660 nm against the blank.

8. Draw a standard curve using A₆₆₀ and the concentration of standard RNA. Calculate the amount in the isolated RNA solution using the graph.