Methodology for Nucleic Acids

Estimation of RNA

A number of reactions have been described for the colorimetric estimation of ribose. Colorimetric procedures suitable for pentose determination have been used for measurement of RNA, and include reactions with orcinol, phloroglucinol, aniline etc. However, the reactions of ribose (Bial’s test) in RNA with orcinol have been widely used.





Principle

The method depends on conversion of the pentose, ribose in the presence of hot acid to furfural which then reacts with orcinol to yield a green color. The color formed largely depends on the concentration of HCl, ferric chloride, orcinol, the time of heating at 100°C etc up to certain maxima.





Materials

» Standard RNA, and Sample RNA Solutions
» Orcinol Acid Reagent
   Add 2mL of a 10% solution (w/v) of ferric chloride. 6 H2O to 400mL of Cone. HC1.
» 6% Alcohol Orcinol
   Dissolve 6gorcinol in 100mL 95% ethanol. Refrigerate in a brown bottle until use. Stable for one month.
» Colorimeter


Procedure
1.
Prepare a standard RNA (50mgRNA/mL) solution in ice-chilled 10mM Tris-acetate, 1mM EDTA buffer (pH 7.2) or any other suitable buffer by dissolving RNA completely.
2.
Dissolve the isolated RNA in the above buffer  solution to  an approximate concentration 50mg/mL.
3.
Prepare a series of tubes containing 0.5mL, 1mL, 1.5mL and 3mL of isolated RNA, 0.5mL, 1mL, 1.5mL and 3mL of 50mg standard RNA/mL.
4.
Make up each tube to 3mL with water. In addition set a blank containing 3mL of water.
5.
Add 6ml of orcinol acid reagent to each tube.
6.
Add 0.4mL 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 20min.
7.
Cool the tubes, and read the absorbance at 660nm 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.


Notes
1.  Dilute using n-butanol if the concentration of the sample is high.
2.  Xylose or adenylic acid can also be used as a standard.
The yield and purity of RNA preparation can be assessed by measuring the absorbance of ultraviolet light by a solution of nucleic acid. A pure RNA solution should give a 260nm: 280nm of 2; one unit of A260 measured in 1cm light path length is equivalent to 5mg/mL.


References
1. Bial, M (1902) Den med Woch 28 253.
2. Ashwell, G (1957) In: Methods in Enzymol 3 (Eds Colowick, S P and Kaplan, N O) Academic Press New York p 87.
3. Sadasivam, S, Radha Shanmugasundaram and Shanmugasundaram, E R B (1975) Arogya J Health Sci 1 125.