Quantitative estimation of RNA using Orcinol reaction

 

Aim

To estimate the quantity of RNA in a given sample by Orcinol reaction using a colorimeter.

Principle

RNA contains the pentose sugar ribose. When RNA is heated with concentrated hydrochloric acid, ribose is converted into furfural. Furfural reacts with Orcinol reagent in the presence of ferric ions to produce a green-colored complex.

The intensity of the green color is directly proportional to the concentration of RNA present in the sample and is measured colorimetrically at 660 nm (red filter).

The reaction involved is represented as:

Ribose Conc. HCl Furfural Orcinol Ferric ions Green complex

Requirements

Apparatus

• Colorimeter
• Test tubes
• Pipettes
• Beakers
• Water bath
• Measuring cylinder
• Test tube stand

Chemicals/Reagents

1. Orcinol Reagent
2. Standard RNA solution
3. Distilled water
4. Unknown RNA sample

Preparation of Orcinol Reagent

Dissolve:

• 1.5 g Orcinol in 500 mL concentrated HCl
• Add 25 mg ferric chloride (FeCl₃)

Store in a brown bottle.

Procedure

1. Label five test tubes as Blank, Standard 1, Standard 2, Standard 3, and Test.
2. Add standard RNA solution in increasing concentrations to the standard tubes.
3. Add the unknown sample to the test tube marked “Test”.
4. Add distilled water to make equal volume in all tubes.
5. Add 3 mL of Orcinol reagent to each tube.
6. Mix thoroughly.
7. Heat all tubes in a boiling water bath for 20 minutes.
8. Cool the tubes to room temperature.
9. Measure the optical density (OD) at 660 nm using a colorimeter against the blank.

Calculation

Prepare a standard graph by plotting:

• RNA concentration on X-axis
• Optical density on Y-axis

Determine the RNA concentration of the unknown sample from the standard graph.

The relationship is:

$\text{RNA concentration} \propto \text{Optical Density (OD)}$

Observation Table

Tube	RNA Amount (µg)	OD at 660 nm
Blank	0	0.00
Standard 1	20	0.12
Standard 2	40	0.25
Standard 3	60	0.38
Test	Unknown	0.30

Using Standard 2 values:

• Standard RNA amount = 40 µg
• OD of Standard = 0.25
• OD of Test = 0.30

Apply the formula:

$\frac{\text{Unknown RNA}}{40}=\frac{0.30}{0.25}$

Therefore,

$\text{Unknown RNA}=\frac{0.30\times40}{0.25}$

$\text{Unknown RNA}=48\,\mu g$

Result

The amount of RNA present in the given unknown sample is 48 µg.

Conclusion

The optical density of the test sample (0.30) lies between Standard 2 and Standard 3, indicating that the RNA concentration is between 40 µg and 60 µg. Hence, the estimated RNA concentration is 48 µg.

Viva-Voce Questions with Answers

1. Why is Orcinol used in RNA estimation?

Orcinol is used because it reacts with ribose sugar present in RNA to produce a green-colored complex, which helps in quantitative estimation of RNA.

2. Which sugar is present in RNA?

The sugar present in RNA is ribose sugar.

3. What is the color produced in Orcinol reaction?

A green color is produced in the Orcinol reaction.

4. At which wavelength is the reading taken?

The optical density is measured at 660 nm using a colorimeter.

5. Why is ferric chloride added to the reagent?

Ferric chloride acts as a catalyst and enhances the formation of the green-colored complex during the reaction.

6. Why is a blank solution necessary?

A blank solution is necessary to calibrate the colorimeter and eliminate errors due to reagents or solvent background absorbance.