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In this article we will discuss about the isolation of RNA with its protocols. Learn how to isolate:- 1. Total RNA from Bacterial Cells 2. Total RNA from Plant Tissues 3. Hot Phenol of RNA from Plant Tissues 4. Messenger RNA.
1. How to Isolate Total RNA from Bacterial Cells?
Protocol:
As usual when working with RNA, always wear gloves, use only freshly prepared and auto- claved material, and use sterile plastic pipettes. Corex tubes and pestle and mortar have to be pretreated with hydrogen peroxide for 30 seconds, rinsed several times with water and incubated for 6 hours at 180°C in an oven.
1. Inoculate bacteria into 10 ml of LB and grow overnight at 37°C.
2. Use 5 ml of this culture to inoculate 50 ml of LB and grow for 2.5 hours at 37°C, to an absorbance of 0.5-0.6 at 600 nm.
3. Centrifuge 1.5 ml of culture in a microfuge tube at 15,000 rpm for 2 minutes.
4. Re-suspend the pellet in 300 μl of 0.85% NaCl.
5. Centrifuge at 13,000 rpm for 2 minutes and discard the supernatant.
6. Re-suspend the pellet in 400 μl of 0.12 M sodium phosphate buffer pH 7.2.
7. Add 50 μl of 5% SDS and 50 μl of proteinase K (0.5 mg/ml).
8. Vortex and incubate at 37°C for 20 minutes.
9. Add 750 μl of 6 M guanidine thiocyanate (GTC).
10. Vortex and centrifuge at 13,000 rpm for 3 minutes.
11. Transfer the supernatant to a fresh tube.
12. Extract sample with acid phenol- chloroform-isoamyl alcohol (25:24:1).
13. Add 0.1 volume of 3 M sodium acetate pH 7.0 and 2.5 volume of cold ethanol.
14. Precipitate RNA at -20°C for 1 hour.
15. Centrifuge at 15,000 rpm for 20 minutes and discard the supernatant.
16. Wash the pellet with 70% ethanol by re-suspending and centrifuge at 15,000 rpm for 10 minutes.
17. Air dry the pellet.
18. Dissolve the pellet in 10-100 μl TE pH 7.0.
Note:
The isolation procedure can be scaled up by increasing the components proportionately. Do not vacuum dry the RNA pellet. It will be very difficult to re-suspend the RNA pellet.
Preparation of Solutions:
2. How to Isolate Total RNA from Plant Tissues?
Protocol:
1. Grind 1 g of tissue with pestle and mortar in liquid nitrogen.
2. Transfer to a 50 ml tube containing 10 ml of extraction solution.
3. Add 1 ml of 2 M sodium acetate pH 4.1.
4. Add 10 ml phenol.
5. Add 2 ml chloroform-isoamyl alcohol (49:1).
6. Mix thoroughly.
7. Shake for 10 seconds and cool on ice for 15 minutes.
8. Centrifuge at 4000 rpm at 4°C for 20 minutes in a microfuge.
9. Transfer aqueous phase to a fresh 30 ml corex glass tube.
10. Add 10 ml isopropanol. Precipitate for 1 hour at -20°C.
11. Centrifuge at 8000 rpm at 4°C for 20 minutes.
12. Dissolve RNA pellet in 3 ml of GTC-ME solution (heat to 60°C to dissolve pellet).
13. Add 3 ml isopropanol and precipitate for 1 hour at -20°C.
14. Centrifuge at 8000 rpm at 4°C for 15 minutes.
15. Re-suspend the pellet in freshly prepared 75% ethanol.
16. Repeat centrifugation, remove supernatant and air dry for 10-15 minutes.
17. Dissolve pellet in 3-5 ml of sterile, freshly autoclaved H2O (heat to 60°C to dissolve pellet).
Preparation of Solutions:
2 M Sodium Acetate pH 4.0 (100 ml):
Dissolve 16.4 g sodium acetate in about 80 ml water.
Adjust the pH to 4.0 with glacial acetic acid.
Make up the volume to 100 ml.
3. How to Isolate Hot Phenol of RNA from Plant Tissues?
Protocol:
1. Grind 1 g leaf material in liquid nitrogen.
2. Add extraction buffer and phenol mixture (80°C) in a 1:2 ratio (1 g leaf material/2 ml) in RNase free tubes, vortex (avoids isolation of genomic DNA).
3. Add 1 volume of chloroform (2 ml). Shake for 30 minutes at room temperature.
4. Centrifuge for 10 minutes at 4000 rpm in a centrifuge.
5. Transfer upper phase to a new tube and add 1/3 volume of 8 M LiCl to bring the final concentration to 2 M and precipitate at 4°C.
6. Centrifuge for 25 minutes at 4000 rpm.
7. Remove supernatant and dissolve pellet in 1 ml of 2 M LiCl.
8. Transfer the solution to a microfuge tube and centrifuge for 5 minutes at 4000 rpm.
9. Wash twice with 70% ethanol (each time centrifuge for 5 minutes).
10. Wash once with 100% ethanol. Air dry for 15 to 30 minutes.
11. Dissolve in 50 μl of distilled water (for 1 g powder).
Preparation of Solutions:
Extraction Buffer-Phenol (1:1 Mixture):
Add equal volume (weight) of phenol to the extraction buffer.
Add 100 mg of hydroxyquinoline.
Before use heat to 80°C and shake.
Fresh phenol—not equilibrated—has to be used and then equilibrated with extraction buffer.
(Use RNase free tubes without any pre-treatment.)
Stock solution
8M Lithium chloride (LiCl)
Dissolve 33.92 g in 70 ml distilled water.
Adjust the pH to 8.0 using NaOH
Make up the volume to 100 ml.
4. How to Isolate Messenger RNA?
Protocol:
1. Bring 1 mg total RNA with distilled water to 600 μl.
2. Incubate at 65°C for 4 minutes.
3. Add 600 μl of 2X binding buffer.
4. Add 40 mg of oligo-dT cellulose equilibrated with 1X binding buffer.
5. Incubate for 15 minutes at room temperature on a rolling incubator (or vortex several times).
6. Centrifuge oligo-dT cellulose and discard the supernatant.
7. Wash twice with 1X binding buffer.
8. Wash twice with wash buffer.
9. Elute with 250 μl of elution buffer at 37°C for 5 minutes.
10. Centrifuge and preserve the supernatant.
11. Elute oligi-dT cellulose again with 250 μl of elution buffer.
12. Centrifuge and preserve the supernatant.
13. Combine the elutes and add distilled water to 600 μl.
14. Add 50 μl of 4M NaCl and precipitate with 2 volumes of cold ethanol.
15. Incubate at -20°C for one hour.
16. Centrifuge at full speed for 10 minutes, wash with 70% ethanol, air dry and dissolve in 20 μl of distilled water.
Preparation of Solutions:
Oligo-dT Cellulose (Type 7, Pharmacia):
Use 40 mg for 1 mg total RNA.
Swell in elution buffer.
Wash four times with elution buffer (30 seconds at full speed)
Equilibrate with 2 to 3 times washing steps using 1X binding buffer.
