EXTRACTION & PURIFICATION OF PLANT RNA
Introduction
A typical cell contains about 10-5 g of RNA, 80-85% of which is rRNA. Most of the
remaining 15-20% consists of a variety of low molecular weight species (tRNA and small
nuclear RNAs). Messenger RNA makes up between 1-5% of total cellular RNA and is
heterogeneous in both size and sequence. This heterogeneous population of molecules
collectively encodes virtually all of the polypeptides synthesized by the cell.
Isolation of pure, undegraded total RNA from plant tissue can be difficult because of high
levels of ribonucleases, pectins and polyphenols that are present in plant material. Another major
problem is the presence and removal of proteins. This problem is commonly solved by lysing the
plant cells in the presence of phenol/chloroform. In these extractions the nucleic acids partition
in the aqueous phase and the proteins partition in the denser phenol/chloroform phase. Both
phenol and chloroform are used because deproteinization is more efficient when two organic
solvents are used instead of one. Phenol alone is not the most efficient solvent for RNA because
it does not completely inhibit RNase activity (ribonucleases are extremely stable enzymes). In
addition, poly A+ RNA is somewhat soluble in phenol. Therefore, both phenol and chloroform
are used in RNA extractions. The final extraction with chloroform also aids in the removal of
traces of phenol from the nucleic acid preparation.
Controlling Ribonuclease Activity
As explained above, it is important to minimize the activity of RNases liberated during cell
lysis. Consequently, it is also important to avoid the accidental introduction of trace amounts of
RNase from other potential sources in the laboratory.
Glassware and plasticware: All laboratory glassware and plasticware should be autoclaved.
Solutions: All solutions should be prepared using sterile dH2O and treated with 0.1% DEPC
(diethyl pyrocarbonate), which is a strong RNase inhibitor. Following the addition of DEPC,
solutions should sit for 12 hours at 37˚C and then be autoclaved for 25 min.
NOTE: DEPC IS SUSPECTED TO BE A CARCINOGEN AND SHOULD BE HANDLED
WITH CARE. DEPC REACTS READILY WITH AMINES AND AS SUCH CANNOT
BE USED TO TREAT TRIS BUFFERS. THESE BUFFERS SHOULD BE MADE IN
DEPC TREATED d H2O AND THEN AUTOCLAVED.
Contamination by workers: A potentially major source of RNase is the hands of the
investigator. Disposable gloves should be worn during the preparation of solutions, the isolation
of RNA and all subsequent manipulations.
1
Buffers
Extraction Buffer:
0.1 M Tris
0.2% (w/v) SDS
pH to 9.0 using HCl
make this buffer in DEPC treated sterile d H2O and autoclave.
3M sodium acetate (pH 5.2)
0.3 M NaCl
Grinding - WEAR GOGGLES
Plant tissue (5-10 g) is ground into a fine powder with a mortar and pestle in liquid nitrogen.
Do not allow the plant material to thaw. If the plant material thaws, it will allow the RNases to
degrade the RNA. The ground powder can be stored at –70˚C in a disposable 50 mL centrifuge
tube.
RNA Isolation - WEAR GOGGLES
1. Add 5.0 mL of extraction buffer and 5.0 mL of buffer saturated phenol into each tube
containing ground plant material. Cover the tube and mix each tube (thoroughly mixing the
contents).
2. Grind the mixture at top speed with the Bio Homogenizer for 45 seconds under the hood.
NOTE THAT PHENOL IS VERY CAUSTIC SO WEAR GLOVES AND EXERCISE
CARE WHEN GRINDING. Repeat the grinding step two more times.
3. Centrifuge the homogenate for 5 min. at top speed in the clinical centrifuge.
4. The centrifugation step should separate the top aqueous phase from the lower phenol phase.
Using a sterile glass pipette, remove the top aqueous phase and place it into a new sterile
disposable centrifuge tube. Do not remove any of the lower phenol phase or cloudy interface.
Place this new tube on ice.
5. Add 5.0 mL of extraction buffer into the tube containing the phenol (not into the new tube, but
into your original tube). Cover this tube, mix the contents well and centrifuge the sample for 5
min. at top speed in the clinical centrifuge. Remove the top aqueous phase with the glass pipette
and add it to the aqueous phase of the first tube. DISCARD THE PHENOL ONLY IN
CONTAINERS SPECIFICALLY MARKED FOR PHENOL DISPOSAL.
6. Add 10 mL of fresh phenol into the tube containing the aqueous phase. Cover the tube, mix
the contents well and centrifuge the mixture for 5 min at top speed in the clinical centrifuge.
Remove the bottom phenol phase with a glass pipette and dispose of the phenol.
2
7. Repeat step 6.
8. Add 10 mL of chloroform into your tube, cover the tube (note that chloroform dissolves
parafilm), mix the contents well and centrifuge the mixture for 5 min. at top speed in the clinical
centrifuge.
9. Remove the top aqueous phase with a sterile glass pipette, but be careful not to disturb the
interface. Place the aqueous phase into a sterile plastic centrifuge tube. Dispose of the
chloroform in the phenol waste container.
10. Add 1.0 mL of 3 M sodium acetate (pH 5.2) and approximately 20 mL of ice cold 95%
ethanol. Cover the tube, mix the contents well and store the tubes overnight at –20˚C.
11. The following day, centrifuge the preparation at 10,000 rpm at 4˚C for 1 hour. Following
centrifugation, discard the supernatant but be careful not to disturb the pellet. Add 0.5 mL of
DEPC treated sterile d H2O into each tube and dissolve the pellet by gentle pipetting.
12. Add 5.0 mL of 3M sodium acetate (pH 5.2) into each tube. This will precipitate the RNA but
not the DNA or tRNA. Cover the tube, mix well and centrifuge the tubes at 8,000 rpm for 20
min.
13. Following the centrifugation, discard the supernatant but be careful not to disturb the pellet.
Resuspend the pellet in 0.5 mL of DEPC treated sterile d H2O and add 4.5 mL of 0.3 M NaCl.
Cover the tube, mix well and centrifuge the mixture at 5,000 rpm for 20 min.
14. This time you want to save the supernatant. Pour the supernatant into a new sterile
centrifuge tube, but be careful not to disturb the pellet. Add 15 mL of ice cold 95% ethanol into
the tube, cover the tube, mix well and store the tube overnight at -20˚C.
15. The following day, centrifuge the preparation at 10,000 rpm for 1 hour at 4˚C. Carefully
discard the supernatant and allow the tube to stand inverted for 10 min. in order to remove any
remaining ethanol.
16. Carefully resuspend the RNA pellet in 50 l of DEPC treated sterile dH2O. Remove the 50
l and place it into a sterile microcentrifuge tube.
RNA Spectrophotometric Analysis
1. Add 2 l of RNA sample to the Nanodrop spectrophotometer in order to determine the
concentration. Be sure to use the RNA program and not the default DNA program.
2. The computer program will calculate the concentration of RNA in ng/l.
3. Calculate the mass of RNA that was isolated.
3
Determination of RNA Quality using Agarose Gel Electrophoresis
1. Make a 1% (w/v) agarose gel containing 0.5 g/mL of ethidium bromide in 1X sodium
phosphate buffer (0.01 M, pH 7.0). This is a10 fold dilution of your 10X phosphate buffer.
2. Add running buffer (0.01 M phosphate buffer, pH 7.0) into the electrophoretic chamber. This
is done by making a 10-fold dilution of the 0.1 M sodium phosphate buffer. Place the
electrophoretic chamber in the cold room.
3. Remove 1 g of your RNA sample and place it into a new microcentrifuge tube. Add enough
DEPC treated sterile dH2O so that the total volume is approximately 10 l.
4. Repeat step 3 except remove 2 g.
5. Add 3-4 l of RNA tracking dye to each sample, mix well, centrifuge for 5 seconds, and load
the RNA into the gel.
6. Run the gel at approximately 50 volts. You will need to circulate the buffer every 30 minutes
in order to avoid degrading your RNA.
7. View the RNA under an ultraviolet light source, photograph the gel, and examine the rRNA
bands. Intact rRNA bands indicate that your RNA is not degraded.
Buffers
10X Phosphate Buffer-0.1M Monobasic sodium phosphate (NaH2PO4.H2O)
a. Make 1 flask of 500 mL each
b. Dissolve the correct amount of monobasic sodium phosphate in 100 mL of dH2O.
c. Adjust the pH to 7.0
d. Bring the volume to 500 mL
e. DEPC treat and autoclave
RNA tracking dye
a. 50% (v/v) glycerol (2.5 mL)
b. 0.25% (w/v) bromophenol blue
c. 0.25% (w/v) xylene cyanol FF
d. Add 2.5 mL of 0.01M phosphate buffer (this is 1X buffer)
e. Label and autoclave
4