Projection Experiment: Test for Ribose in RNA

Demonstration of Microscale Projection Experiments - Chemistry en miniature

Test for Ribose in RNA

Orcin Reaction

Peter Keusch

German version

Material:
Cells of the thymus gland of the calf

Chemicals:
saturated solution of sodium dodecyl sulfate
conc. hydrocloric acid
1 M sulfuric acid
RNA from yeas
DNA from herring sperm
ORCIN-REAGENT: 0.1 g Orcin and 0.05 g FeCl₃, dissolved in 50 mL conc HCl

Hazards and safety precautions:

Conc. hydrochloric acid is extremely corrosive. Inhalation of vapour can cause serious injury. Ingestion may be fatal. Liquid can cause severe damage to skin and eyes.

Safety glasses and protective gloves should be worn. The experiment should be performed in a portable fume hood giving all-round visibility!

Experimental procedure:

Three test tubes are set up as described below.

Test tube 1 0.5 mL of DNA solution ( a few mg of DNA dissolved in 1 M H₂SO₄ )
Test tube 2 0.5 mL of RNA solution ( a few mg of RNA dissolved in 1 M H₂SO₄ )
Test tube 3 0.5 mL of DNA hydrolyzate from thymus

The samples in T1, T2 and T3 are each mixed with 1.5 mL of ORCIN REAGENT. Afterwards they are placed for 1-2 minutes in a boiling water bath.

Results:

Test tube 1 grey-violet
Test tube 2 green
Test tube 3 green
Turbidity caused by insoluble portions of the hydrolyzate

Discussion:

· The orcin reaction is a test for the presence of ribose in RNA. The reaction depends on the conversion of ribose to an aromatic aldehyde (furfural) which then reacts with Orcin
(3,5-dihydroxy toluene) to give a blue-green condensation product (test tube 2 and 3).

· 2-desoxyribose is converted in acidic medium to 3-acetylacrolein, which reacts with 3,5-dihydroxy toluene (0rcin) to form an aldehyde-phenol condensation product. Oxidation yields a purple dye (test tube 1).

It is shown that the orcin reaction may be applied for simultaneous determination of the DNA content (by extinction at 600 nm after 2 min of the orcin reaction) and RNA in biologic materials. By calculating for DNA standard solutions the value of the ratio between the extinction at 665 nm after 15 min to the extinction of 600 nm after 2 min of the orcin reaction it is possible to increase specifiaty of the orcin method for determination of the RNA content.

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