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BACKGROUND
There are different kinds of methods and reactions that help in describing the colorimeter determination of ribose. The colorimetric process that is suitable for pentose determination have been utilised for measuring RNA and involves reactions with orcinol, aniline, phloroglucinol etc. But one of the widely used reactions is those of RNA with Orcinol. So, the basic objective of this test is to estimate RNA by orcinol method.
Principle: The method usually involves the pentose conversion and ribose in the presence of hot acid to furfural that again reacts with orcinol to form a green colour. The colour that is formed largely depends on HCL concentration, ferric chloride, orcinol and the time of heating at 100oC to some extent.
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REQUIREMENTS
Chemicals: Standard RNA
Sample RNA solution
Orcinol Acid reagent- 2ml of 10% of ferric chloride.6H2O +400ml conc. HCl
6% alcohol orcinol – 6g Orcinol in 100ml 95% ethanol. Keep this solution in a bottle till use.
Others: Colorimeter.
Test tubes
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PROCEDURE
At first prepare standard RNA (50 microgram RNA /ml) solution in 10 mM Trisacetate (chilled), 1 mM EDTA buffer or any such buffer and RNA should dissolve perfectly.2 Now make ready a series of tubes with 0.5 ml, 1 ml, 1.5 ml, and 3 ml of isolated RNA and another series of 0.5 ml, 1 ml, 1.5 ml and 3 ml of 50 microgram of standard RNA/ml.1 Now add water to make total volume of each tube to 3 ml. In another test tube add 3 ml of water to keep it as blank.3 Now take 6 ml of orcinol acid reagent and add to each tube. Again add 0.4 ml of 6% alcoholic orcinol to every tube. Now shake every tube so that everything is mixed up very perfectly and keep it in boiling water bath for about 20 min. Allow the tubes to cool down and then take absorbance reading at 660 nm against the blank. Now draw a standard curve using A660 and the concentration of standard RNA. Calculate the amount in the isolated RNA using the graph.
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CONCLUSION
It is one of the specific methods for determination of RNA in the presence of DNA and proteins.
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REFERENCES
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Gurin, S., and Hood, D. B., J. Biol. Chem., 139, 775 (1941); 131, 211 (1939).
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Dische, Z., Proc. Sot. Ezp. Biol. and Med., 66, 217 (1944).
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Mejbaum, W., 2. physio2. Chem., 268, 117 (1939). 6. Schneider, W. C., J. Biol. Chem., 181,293 (1945).
