RNA estimation by Orcinol method

Introduction

The colorimetric method suitable for pentose determination have been utilize for measuring RNA and involve reaction with orcinol, aniline, phloroglucinol etc. One of the widely used reaction is those of RNA with orcinol commonly used for estimation purpose.

Principle

This is a general method for estimation of pentoses. Acid hydrolysis of RNA releases ribose which in the presence of strong acid undergoes dehydration to yield furfural. Orcinol, in the presence of ferric chloride as a catalyst, react with furfural producing a green colour compound with absorbance maxima at 665 nm. DNA gives a positive reaction with orcinol test.

Requirements

1. Standard RNA solution (250 μgm/ml)
2. Unknown RNA sample solution
3. Orcinol reagent
4. Distilled water
5. Spectrophotometer
6. Boiling waterbath
7. Pippet, Test tube, Graph paper.

Procedure

Arrange the tubes as show in table to prepare standard curve and add the reagent according to the table. Similarly prepare tubes for sample and Proceed as per table.

Take OD at 665 nm and calculate the RNA concentration from standard graph of sample.

Reference

Jain A., Jain R., Jain S. (2020) Estimation of RNA Using Orcinol Method. In: Basic Techniques in Biochemistry, Microbiology and Molecular Biology. Springer Protocols Handbooks. Humana, New York, NY.