RNA ISOLATION FROM PLANT TISSUE
Modified from Hensgens and Van Os-Ruygrok, Rockefeller Foundation Report.
I. Isolation Buffer
A. Stock solutions
1. 5X tri-iso-propylnaphthalene disulfonic acid
a. Transfer 5 g into ~75 ml dH20
b. Heat with stirring to get into solution
c. Bring volume to 100 ml with dH20
d. Filter through Whatman #1 filter paper
e. Store in plastic bottle covered with foil at room temperature
2. 10X 4-amino-2-hydroxybenzoic acid
a. Transfer 20 g into ~75 ml dH20
b. Stir to get into solution
c. Bring volume to 100 ml with dH20
d. Filter through Whatman #1 filter paper
e. Aliquot and store at -20o C
B. Preparation of working solution (10 ml--enough for 5 g tissue--scale as needed)
1. Mix:
0.5ml 1 M Tris-HCl
1.0 ml 0.5 M EDTA
1.0 ml 5 M NaCl
2 ml 5X tri-iso-propylnaphthalene disulfonic acid stock
1 ml 10X 4-amino-2-hydroxybenzoic acid stock
2 ml 20% SDS stock
7 μl β-mercaptoethanol
1.89 ml RNase-free dH20
2. Filter through Millipore GVWP membrane
3. Add 0.6 ml 2-butanol
4. Check pH of final solution (pH 8.5 is desired)
C. Final concentrations
50 mM Tris-HCl
50 mM EDTA
0.5 M NaCl
10 mM β-mercaptoethanol (0.698 ml/l)
1% (w/v) tri-iso-propylnaphthalene disulfonic acid
2% (w/v) 4-amino-2-hydroxybenzoic acid
4% (w/v) SDS
6% (v/v) 2-butanol
II. DEPC Treatment
A. Glass- and plasticware required for RNA preps
1. Waring blender jar
2. Glass stirring rod (1/prep.)
3. Beakers: 100 and 250 ml (1 of each/prep.)
4. Magnetic stir bars (2/prep.)
5. Side-arm flask: 125 or 250 ml (1/prep.)
6. Glass bottles: 250 ml (2/prep.)
7. Pasteur pipettes (several/prep.)
8. 10-ml pipettes (3/prep.)
9. Glass funnel (1/prep.)
10. 30-ml Corex tubes (number needed varies; 18 required for 20-g prep.)
11. Plasticware: 1.5-ml Eppendorf tubes, Pipetteman tips
B. DEPC treatment of glass- and plasticware
1. Mix DEPC (0.05%) in 4 l of dH20 in plastic beaker; place in fume hood
2. Place blender jar and other glassware to be treated in beaker
3. Cover top of beaker with foil; leave in hood overnight (O.N.)
4. Remove glassware, cover openings with foil and autoclave
5. Cover plastic beaker with foil, autoclave, pour contents down drain
C. DEPC treatment of water and solutions
1. Add the appropriate amount of DEPC (0.05%) to bottles, shake
2. Allow to stand O.N. in hood
3. Autoclave
4. Note: DO NOT DEPC-treat solutions containing Tris or SDS!
III. Equilibrating Phenol
A. Melt the phenol in a warm water bath or microwave carefully
B. Add 1X volume of 1 M Tris, pH 8.0-8.5; mix by shaking carefully
C. Let the mixture stand at 4o C until the phases separate
D. Remove the aqueous (upper) phase from the bottle
E. Add 1X volume of 0.1 M Tris, pH 8.0-8.5
F. Mix carefully and allow the phases to separate at 4o C
G. Measure the pH of the aqueous phase (it should be >7.6)
H. Cover bottle containing the equilibrated phenol with foil and store at 4o C
I. If the pH is <7.6, repeat steps E-G until satisfactory pH is obtained
J. Note: with each successive equilibration step, the volume of the phenol will
decrease; therefore, excessive equilibration steps should be avoided.
IV. Isolation Procedure
A. Freeze the tissue in liquid nitrogen (LN2), transfer to a blender pre-chilled with LN2 and blend to
a very fine powder.
B. Transfer the powder to an RNase-free beaker and let the LN2 boil off. Add isolation buffer (2X
the tissue weight) and let thaw.
C. Grind the tissue in isolation buffer with Polytron.
D. Stir the mixture on a magnetic stir plate for 10 min.
E. Transfer the slurry to an RNase-free glass bottle (with Teflon-lined cap) and add an equal
volume of equilibrated phenol. Shake the bottle 10 min.
F. Transfer the mixture, using an RNase-free glass funnel, to RNase-free 30-ml Corex tubes. Spin at
10K for 10 min.
G. Transfer the aqueous (upper) phase to the glass bottle and repeat the phenol extraction.
H. Transfer the aqueous phase from the second phenol extraction to a fresh RNase-free bottle. Add
an equal volume of chloroform and shake 10 min.
I. Transfer the mixture to fresh RNase-free 30-ml Corex tubes and spin at 10K for 10 min.
J. Transfer the aqueous phases to weighed Corex tubes and determine the volume in each.
K. Add 0.1 volume 3 M NaOAc and 2.5 volumes 95% ethanol to each tube. Cover and invert the
tubes to mix; store O.N. at -20o C
L. Spin the tubes at 10K for 10 min. Remove the supernat. and wash the pellets twice with 70%
ethanol. Dry the pellets (open tubes on side on benchtop).
M. Dissolve the pellets in a total of 6 ml TES*. Combine the samples and add 2 ml 8 M LiCl to the
total vol. (final concen. is 2 M). Mix well and incubate for at least 8 hr. at 4o C.
N. Spin the tube at 10K for 15 min. Remove the supernatant and wash the pellet three times with
70% ethanol. Dry the pellet.
O. Dissolve the pellet in 1 ml TE or water (this may take some heating).
*
TES: 50 mM Tris-HCl, 10 mM EDTA (pH 8.0), 0.1% (w/v) SDS.
V. RNA Quantitation
A. Measure the A260 of a 1:100 to 1:1000 dilution of the final RNA solution
B. [RNA]=(A260)(dilution factor)(40)
C. Calculate the yield of RNA per gram fresh weight of tissue extracted
VI. Electrophoresis of RNA
A. 5X buffer (see Sambrook et al., 1989; p. 7.43 for add'l. info.)
1. Preparation
a. Make 800 ml of 50 mM NaOAc
i. 13.3 ml RNase-free 3 M NaOAc
ii. 786.7 ml RNase-free dH20
b. Add 20.6 g MOPS
c. pH to 7.0 with NaOH
d. Add 10 ml RNase-free 0.5 M EDTA
e. Take volume to 1 l
f. Filter-sterilize; transfer to sterile bottle
2. Final concentrations
0.1 M MOPS
40 mM NaOAc
5 mM EDTA
B. Loading buffer
1. Preparation
a. Mix:
5 ml glycerol
20 μl 0.5 M EDTA
25 mg bromophenol blue
25 mg xylene cyanol FF
4.98 ml dH20
10 μl DEPC
b. Autoclave
2. Final concentrations
50% glycerol
0.1 mM EDTA
0.25% bromophenol blue
0.25% xylene cyanol FF
C. 1% agarose gel (this recipe for 75-ml gel)
1. Add 0.75 g agarose to 60 ml dH20
2. Add 15 ml 5X buffer
3. Microwave the mixture to melt the agarose
4. Pour into leveled molding tray with comb in place
D. Running buffer
Mix: 187.5 ml 5X buffer and 750 ml dH20
E. Samples and markers
1. Mix:
RNA sample and dH20 to final volume of 4.5 ul
2 μl 5X buffer
3.5 μl formaldehyde
10 μl formamide
2. Heat in 65o C water bath for 5 min.
3. Quench tubes on ice
4. Add 2 μl loading buffer
5. Spin tubes briefly
6. Load into wells of the gel