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R.N.

Presented by-damini Bsc.M.L.T-3rd year Moderator-Dr.Ramneet.

Ribonucleic acid (RNA) is one of the three major macromolecules (along with DNA and proteins) that are essential for all known forms of life.
RNA is made up of a long chain of components called nucleotides. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate group.

Structure of RNA
Single stranded. Ribose Sugar.

5 carbon sugar.
Phosphate group. Adenine, Uracil, Cytosine, Guanine.

Demonstration
Ribonucleic acid methods are not specific. They depend upon the non specific property of their affinity for basic dyes. The basophilia is produced by the acidic,i.e phosphate groups in the nucleic acid.

Methyl green-pyronine method


Methyl green is the most selective basic dye for nucleic acids. It is an impure dye and chloroform extraction is necessary to remove the crystals and methyl violet impurities.

When treated in this way and used at an acidic pH,it appears to be specific for DNA.

This is due to spatial alignment of phosphate radicals of the DNA to the amino group on the methyl green molecules. Pyronine binds to the RNA by the normal electrostatic mechanism,i.e.cationic dye to anionic nucleic acid. The selectivity of pyronine to RNA is not high,but with careful differentiation the use of pyronine produces acceptable results.

pyronine is not specific for RNA, cartilage, osteoid, keratin, eosinophil granules and mast cell granules will also stain. STAINING SOLUTION 2% aqueous methyl green. 2%aqueous pyronine Y. acetate buffer pH 4.8. glycerol.

TECHNIQUE
Bring section to water. Rinse in acetate buffer. Staining soln.25 min. Rinse in buffer. Blot dry. Dehydrate,clear and mount.

RESULTS
DNA-green blue. RNA-red. This method is particularly useful for the identification of plasma cells that have a high conc. of RNA related to the synthesis of immunoglobulin molecules.

HITCHCOCK-EHRICH TECHNIQUE
This method has the advantage of being quick and simple. It uses malachite green and acridine red.
STAINING SOLUTION

Malachite green
Acridine red

0.3 gm in 15ml D.W


0.9 gm in 45ml D.W

TECHNIQUE
Bring section to water. Staining soln.1-5 min. Wash in water. Dehydrate,clear and mount.

RESULTS
DNA-blue green. RNA-red.

GALLOCYANIN-CHROME ALUM METHOD


Gallocyanin forms a red cation lake with chromium potassium sulphate. The dye lake combines with phosphate groups of nucleic acids to form a tissue-mordant dye compound.

The method demonstrates both the nucleic acids and the staining is progressive.

STAINING SOLUTION
Chrome alum D.W 5gm 100ml

Chrome alum is dissolved in D.W, gallocyanin added and the soln. heated until it boils.Boil for 5 min.,cool at room temp.

TECHNIQUE
Bring section to water. Stain in gallocyanin chrome alum soln.(18-48 hrs.). Dehydrate,clear and mount. RESULTS DNA,RNA-grey blue.

since DNA and RNA appear of the same colour hence extraction techniqueis necessary to isolate a specific nucleic acid.

EXTRACTION TECHNIQUE
These techniques remove or denature nucleic acid,leaving other tissue structures unaffected.

Extraction can be enzymatic or chemical. ENZYMATIC EXTRACTION use deoxyribonuclease and ribonuclease.

RIBONUCLEASE-the enzyme splits RNA into its component nucleotides and is produced from fresh beef pancreas. A crude source of the enzyme is found in saliva. Mercuric chloride and potassium dichromate fixation of tissue inhibit ribonuclease digestion. CHEMICAL EXTRACTION-it uses various acids to extract nucleic acids.

ENZYME EXTRACTION OF RNA


SOLUTION Ribonuclease in D.W TECHNIQUE Bring test and control sections to water. Place test and positive control slide in ribonuclease soln. Place negative control in D.W,at 37 C for 1 hr. Wash in D.W. Apply methyl green pyronine method. Another method for RNA extraction is with perchloric acid.

FLUORESCENT METHOD FOR RNA DEMONSTRATION


Fluorescent dyes that can be used are acridine orange and acriflavine. Alcohol is the choice of fixative. Fluorescent method is mainly helpful in detection of malignant cells. Rapidly growing malignant cells,have an increased cytoplasmic RNA content and this has afforded a means by which malignant cells can be detected.

ACRIDINE ORANGE TECHNIQUE


The technique requires a pH of 6.0 for the differential staining of RNA and DNA.

SOLUTIONS Acridine orange soln.-0.1% aq acridine orange.


Buffer-phosphate buffer pH6.0

Differentiator-0.1M calcium carbonate.

TECHNIQUE
Bring section to water. Rinse in 1%acetic acid for a few seconds then two changes of D.W. Stain in the diluted acridine orange soln.,3min. Rinse in buffer. Differentiate in 0.1M calcium chloride soln.,1/2min. Wash in phosphate buffer.

RESULTS
DNA-yellow green. RNA-red.

The technique is no more popular due to the impermanence of the preparations. The technique cannot be successfully applied to formalin and Bouins soln. fixed tissues.

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