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In this article we will discuss about the requirements and procedure for isolation of RNA using Phenol-SDS method.
SDS dissociates the ribonucleoprotein complex into RNA and protein, deproteinised by phenol and the free RNA left in aqueous solution is precipitated in cold after adding alcohol.
Requirements:
Reagents:
1. Extraction buffer-pH 9.0:
Tris HCl (0.1 M): 1.21 g
NaCl (0.075M): 0.44 g
EDTA Na2 (0.005M): 0.19 g
Distilled water to make up vol. to: 100.00 ml
2. SDS 10% (W/V) in distilled water.
3. Ether.
4. Ethanol.
5. Freshly redistilled Phenol.
6. Liquid N2.
7. Bench top refrigerated centrifuge.
8. 4°C refrigerated chamber.
9. Magnetic stirrer.
10. Mortar and pestle (ice cold).
11. Centrifuge tubes.
12. Vortex mixer.
13. Plant material: leaves/callus.
14. DEPC treated water.
Procedure:
1. To avoid contamination by any nuclease, all glasswares should be baked at 120°C overnight. All preparations should be carried out in cold condition (0—4°C). Care should also be taken to treat all glasswares, mortar and pestle with DEPC treated water. All solutions should be prepared in DEPC water to prevent RNAse activity.
2. Freeze 0.5—5.0 g of plant material and grind with liquid N2 in a pre-cooled mortar and pestle first to a powder and then to a paste and extract in 10 volumes of extraction buffer (pH 9.0).
3. Centrifuge the homogenate at 2000 rpm for 5 minutes.
4. Transfer the supernatant to a volumetric flask and stir with 0.1 volume of 10% SDS for 2-3 minutes.
5. Add an equal volume of buffered phenol (buffered phenol=freshly redistilled phenol saturated overnight with 100 mMTris- HCl, pH 8.5).
6. Centrifuge at 5000 rpm for 5 minutes.
7. Collect the upper aqueous phase into a separate flask.
8. Add an equal volume of extraction buffer to the lower and interphase, shake for 5 minutes and centrifuge.
9. Pour the aqueous phase mentioned in step 7 into the flask and stir with an equal volume of buffered phenol (step-5) for five minutes.
10. Repeat steps 8 and 9 for at least five times or until the interphase shows no proteins.
11. Collect the upper aqueous phase containing RNA and dissolve in it 250 mg NaCl, add two volumes of cold 95% ethanol.
12. Leave the flask at -20°C overnight, for RNA to precipitate.
13. Centrifuge at 2000 rpm for ten minutes.
14. Wash the pellet of RNA with 70% ethanol, ethanol: ether (1:1) and finally with ether.
15. Gently dry the pellet in vacuum for few minutes.
16. Dissolve RNA completely in elution buffer for further analysis by vortexing.
Elution buffer (pH 7.5):
Tris 0.01M: 0.12 g
EDTA Na2 0.001 M: 0.04 g
SDS 10%: 0.50 ml
Distilled water to make: 100.00 ml
17. Dilute 20 µl aliquot to 2 ml with buffer. Read absorbance using 1 cm light path cuvette at 260 nm in a spectrophotometer. One (OD260) unit is assumed equivalent to 40 µg RNA/ml.