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  Section: Plant Lab Protocols
 
 
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Methodology for Nucleic Acids

 
     
 
Isolation of ribonucleic acid
 
Ribonucleic acid (RNA) is the transcriptional product of DNA. The total RNA includes three classes - ribosomal, messenger and transfer RNA. Ribonucleic acid occurs as ribonucleoprotein particle in intact cells. There are a number of methods described for the extraction of total RNA. The method selected largely depends upon the source material and the experimental use for which RNA is extracted. Among the two common methods used with plant materials, the phenol-chloroform method is used to recover the total RNA intact. The phenol-SDS method described below is useful for quantitative recovery.
 
 



Principle

The ribonucleoprotein complex is dissociated by SDS into RNA and protein, deproteinized by phenol and the free RNA left in aqueous solution is precipitated in the cold after adding alcohol.
 
 
Materials
Centrifuge (bench top)
Magnetic stirrer
Cold room (4°C)
Phenol (freshly redistilled)
Extraction buffer (pH 9.0)
   Tris-HCl (0.1M)                 1.21g
   NaCl (0.075M)                  0.44g
   EDTA Na2 (0.005M)          0.19g
   Water to                              100mL

Ethanol
SDS 10% (w/v in water)
Ether
All glassware should be baked at 120°C overnight. Contamination with any nuclease should be strictly avoided.
 
 


Procedure
All operations should be done at 0-4°C.
1.
Freeze 0.5-5g of the material in a mortar and pestle with liquid N2, grind to a fine powder, then to a paste and extract in 10 volumes of extraction buffer.
2.
Centrifuge the homogenate at 2,000 x g for 3 min.
3.
Transfer the supernatant to a volumetric flask and stir with 0.1 volume of 10% SDS for 2-3 min.
4.
Add an equal volume of buffered phenol (freshly redistilled phenol saturated overnight with 100mM Tris-HCl pH 8.5)
5.
Partition the content by centrifuging at 2,000 x g for 5 min and collect the upper aqueous phase into a separate flask.
6.
Shake the lower and interphase again with an equal volume of extraction buffer for 5 min and centrifuge.
7.
Combine the aqueous phase with the first one (step 5) and stir with an equal volume of buffered-phenol for 5 min.
8.
Repeat the extraction and centrifugation steps at least five times or until the interphase shows no proteins.
9.
Finally, collect the upper aqueous phase containing RNA, dissolve in it about 250mg NaCl, add two volumes of cold ethanol (96%) and leave the flask overnight at -20°C for RNA precipitation.
10.
Collect RNA by centrifugation at 2,000 x g for 10 min. Wash the pellet (RNA) with 70% ethanol, ethanol, ethanol: ether (1:1 v/v) and finally with ether. Dry the pellet gently ‘in vacuo’ for a few minutes.
11.
Dissolve the RNA completely in elution buffer (see isolation of mRNA) for further analysis by vortexing.
12.
Dilute 2mL aliquot to 2mL with buffer and read the absorbance using lcm light path cuvette at 260nm in a spectrophotometer. One A260 unit is assumed equivalent to 50mg RNA/mL. Otherwise, the RNA content is estimated colorimetrically (see estimation of RNA).
 
 


Notes
1.
All the glassware and solutions should be sterile. Any contaminating RNase is inactivated by rinsing the glassware with 1 % diethyl pyrocarbonate solution.
2.
Use freshly distilled colourless phenol. It can be stored frozen in small aliquots in brown-colored bottles. Wear gloves while handling phenol.
3.
A variety of extraction medium, chelating agents, deproteinizing agents etc are used for the extraction of RNA.
4.
The denatured proteins gather at the interphase after low speed centrifugation which should be discarded
5.
The volume of aqueous phase will be drastically reduced if the phenol is not fully saturated.
6.
Any contaminating DNA will appear just like cotton wool during ethanol addition.
7.
Deproteinization using phenol leads to the loss of poly (A)-tail of mRNA considerably.
8.
Phenol: Chloroform (1:1) mixture is an effective deproteinizing agent that retains the poly (A) tail in mRNA intact.
 
 


References
1.  Brawerman, G (1974) In: Methods in Enzymol 30 (Eds Moldave and Grossman, L) Academic Press New York p 605.
2.  Perry, R P, La Torre, J, Kelley, D E and Greenberg, J R (1972) Biochem BiophysActa 262 220.
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