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Orcinol Method of RNA Estimation

In this Orcinol method of RNA estimation post we briefly summarise about: principle, reagents requirements, orcinol method procedure, result, application and limitations of Orcinol method.

Orcinol Method of RNA Estimation

Orcinol method is a general reaction for pentoses. The orcinol reagent reacts with pentose groups in the backbone of the RNA molecule. Many downstream applications require high-quality RNA. The quality criteria for downstream applications might vary greatly, and RNA that is unacceptable for one application may produce completely acceptable results in another.

Due to the widespread availability of RNases in the environment, RNA is very vulnerable to destruction. The most frequent approach for RNA measurement will be discussed in this post.

Principle

This is a general pentose reaction that relies on the production of furfural when the pentose is cooked in strong hydrochloric acid. In the presence of ferric chloride as a catalyst, orcinol interacts with furfural to produce a green colour that can be detected at 665 nm.

Requirements

Materials

  1. Pipette
  2. Test tube
  3. Test tube stand
  4. Vortex
  5. Spectrophotometer
  6. Water bath

Reagent

  1. Perchloric Acid
  2. Ferric Chloride
  3. Hydrochloric Acid
  4. Ethanol
  5. Sodium Chloride
  6. Sodium Citrate
  7. Standard RNA

Preparation

Standard RNA: Standard RNA solution- Dissolve 200µg/ml Standard RNA in 1 N perchloric acid/buffered Saline.

Orcinol Reagent: Dissolve 0.1g of ferric chloride in 100 ml of concentrated HCl and add 3.5 ml of 6% w/v orcinol in alcohol.

Buffered Saline: Prepare 800 mL of distilled water in a suitable container. Add 8 g of Sodium chloride, 200 mg of Potassium chloride, 1.44 g of Disodium phosphate, and 245 mg of Monopotassium phosphate to the solution. Adjust solution to desired pH (typically pH ≈ 7.4).

Procedure

  1. Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labelled test tubes. Pipette out 1 ml of the given sample in another test tube. Make up the volume to 1 ml in all the test tubes.
  2. A tube with 1 ml of distilled water serves as the blank. Now add 2 ml of orcinol reagent to all the test tubes including the test tubes labelled ‘blank’ and ‘unknown’. Mix the contents of the tubes by vortexing / shaking the tubes and heat on a boiling water bath for 20 min.
  3. Then cool the contents and record the absorbance at 665 nm against blank. Then plot the standard curve by taking concentration of RNA along X-axis and absorbance at 665 nm along Y-axis. Then from this standard curve calculate the concentration of RNA in the given sample.

Calculation

Orcinol Method of RNA Estimation

Concentration of RNA present in the given unknown sample is ———µg/2 mL.

Further Readings

Reference