Isolation of Plant mRNA
Procedure
1. Use young plants, about 4 inches tall or 6 to 7 days old for barley. Cut about 10 grams of leaves and rinse briefly with sterile ddH2O containing 0.1% (v/v) Diethylpyrocarbonate, DEPC (Sigma). For DEPC treatment, see Maniatis. Blot dry leaves.
2. Add to large acid-washed, sterile, DEPC-treated mortar chilled with liquid nitrogen. Grind for several minutes, adding more liquid nitrogen as needed.
3. Add ground leaves to 30 ml GITC mixture, and stir to mix. Incubate at 65°C for 10 minutes with occasional swirling. The viscosity of the solution should greatly increase.
4. Pass the solution through an 18-gauge or smaller needle several times to reduce viscosity.
5. Spin in a warm rotor (i.e. at room temperature) at 10,000 rpm for 30 minutes in SS34 rotor. Collect supernatant.
6. Add CsCl at 0.2 g/ml. Place into clean 25 ml polyallomer
tubes. Underlayer 1/4 of tube (about 2 ml) with 5.7 M
CsCl (5.7 M CsCl + 0.1 M NaEDTA, DEPC-treated and filtered
through 0.45 mm filter), and spin in SW28.1 rotor for 24 hours
at 20°C at 24,000 rpm (described in Biochem. (1979) 18:5295-5299).
SW40 rotor, 20°C, 30,000 rpm, 12 hours
7. Decant from top, leaving the pellet. Keep tubes inverted. Dry walls of tube with a Kimwipe, and dissolve pellet in a total of 5 to 10 ml of TESB.
8. Heat to 55°C to dissolve pellet (may require 20 minutes). Add Proteinase K at 200 mg/ml (20 ml of 10 mg/ml stock per ml of TESB), and hold at 55°C for 30 minutes. 37°C?
9. Add phenyl methane sulfonyl fluoride (PMSF) to 0.1 mM,
mix, and cool to room temperature.
Use 1.0 ml of 100 mM PMSF per ml of TESB (100 mM
PMSF = 17.419 mg in 1 ml of 100% isopropanol,
store at -20°C) Note: PMSF is dangerous.
10. Extract with phenol, phenol-chloroform, chloroform, and diethyl ether. Dry at 50°C for 10 minutes or under a vacuum to remove remaining traces of ether. Add one tenth volume of 3 M sodium acetate, mix, chill to 0°C, and add 2.5 times the volume of ethanol. Allow to precipitate overnight at -70°C.
11. Pellet the RNA by spinning in SS34 rotor for 10 minutes at 15,000 rpm. Or 25 minutes at 8000 rpm in Falcon 2059 tube (with rubber adaptor). Pour off supernatant.
12. Dissolve pellet in 5 ml 1´ sodium citrate buffer. If necessary, place at 65°C for a few minutes to aid in dissolving pellet. Measure the concentration of total RNA. Use 20 ml sample + 480 ml 1´ Sodium citrate, in 0.5 ml cuvette.
13. Heat RNA to 65°C for 5 minutes and add oligo-dT cellulose (Type 7 from Pharmacia, 27-5543-02) that has already been wetted in 5 ml of 1´ buffer. Mix gently with for 2 hours or more at room temperature. 1 hour is ok. Different people use different amounts of cellulose; Maniatis recommends 0.3 g dry cellulose for each 0.5 mg of RNA. High. 0.25 g/1.8 mg. Used 250 mg/840 mg.
14. Load cellulose and RNA onto a column, e.g. 1 or 3 cc plastic syringe or a DEPC-treated, siliconized glass column. The column should run very slowly and cannot run dry, always maintain a small amount of liquid above the cellulose.
14a. Run sample through the column, followed by 1´ buffer until about 10 ml total has gone through. Run this solution through again, to make sure all of the poly-A RNA is bound.
15. Continue to run 1´ buffer or rinse buffer through the column until the A260 is less than 0.05. Usually will have to run about 5 to 10 ml of buffer through the column.
16. Elute poly-A mRNA with 3 ml elution buffer at 55°C to 65°C.
17. Regenerate column by rinsing with 10 ml ddH2O, 10 ml NaOH-EDTA solution, and 10+ ml of 1´ sodium citrate buffer. The final pH after washing should be 7.6. Store column in 95% ethanol at -20°C with parafilm over the top and bottom.
18. Add 0.3 (0.1?) volume of 3 M sodium acetate and 2.5 volumes of ethanol. Allow to precipitate overnight. Thaw and spin at 15,000 rpm for 10 minutes. Wash pellet with 70% ethanol and dry in vacuum for 5 to 10 minutes.
19. Dissolve pellet in about 100 ml of TESB. Add 900 ml of 100% DMSO (dimethylsulfoxide) and mix well. Add 100 ml 2´ sodium citrate buffer, mix, and heat to 55°C to 65°C for 5 to 7 minutes. Reduces 2° structure.
20. Add 11 ml of 55°C to 65°C 1´ buffer. Add mixture to 10 ml of Oligo-dT in 1´ sodium citrate buffer. Mix for 2 hours at room temperature. Load column and catch elute. Heat to 65°C for 5 minutes and again run through column.
21. Rinse column with 10 ml of 1´ buffer or rinse buffer until A260 is less than 0.05.
22. Elute with 3 ml of heated elution buffer. Ethanol precipitate. Dissolve in ddH2O or desired buffer, but remember that EDTA will inhibit reverse transcriptase. Long-term storage is possible under 70% ethanol at -70°C.
23. Expect A260/A280 of 2.0 to 2.2. Calculate concentration of poly-A mRNA.
24. Run on a denaturing gel to assess the purity. See Maniatis for glyoxal gels.
GITC
100 g Guanidium isothiocyanate 100 ml ddH2O 10.6 ml 1 M Tris-Cl (pH 7.6) 10.6 ml 0.5 M Na2 EDTA
Stir overnight at room temperature and heat to 65°C for 10 minutes. May need to spin at 3000g for 10 minutes at 20°C to remove residue. Add 21.2 ml 20% Sarkosyl or SDS and 2.1 ml b-mercaptoethanol. Bring volume up to 212 ml with sterile water, and filter through 0.4 mm Millipore filter. Store at 4°C, wrapped in foil. Must not let final concentration of GI go below 2.2 M
1X Sodium Citrate - NaCl Buffer, 10 ml 2X Sodium Citrate - NaCl Buffer, 10 ml
50 mM Na citrate (pH 7.6) 0.5 ml of 1 M stock 0.5 M NaCl 1.0 ml of 5 M stock 1 mM EDTA 20 ml of 0.5 M stock 8.48 ml sterile H2O Final volume 10 ml
Rinse Buffer, 10 ml
100 mM Na citrate (pH 7.6) 1.0 ml of 1 M stock 1.0 M NaCl 2.0 ml of 5 M stock 2 mM EDTA 40 ml of 0.5 M stock 6.96 ml sterile H2O Final volume 10 ml Elution buffer, 10 ml
50 mM Na citrate (pH 7.6) 0.5 ml of 1 M stock 0.1 M NaCl 200 ml of 5 M stock 1 mM EDTA 20 ml of 0.5 M stock 9.3 ml sterile H2O 10 ml final volume NaOH-EDTA, 10 ml
10 mM Na citrate (pH 7.6) 100 ml of 1 M stock 1 mM EDTA 20 ml of 0.5 M stock 9.88 ml sterile H2O Final volume 10 ml TESB, 20 ml
0.1 M HaOH 40 mg of NaOH 5 mM EDTA 100 ml of 0.5 M stock 9.86 ml sterile H2O Final volume 10 ml
To make: 20 ml 50 ml 10 mM Tris-Cl (pH 7.6) 200 ml 500 ml 1 M Tris 1 mM EDTA 40 ml 100 ml 0.5 M EDTA 0.1% SDS 100 ml 250 ml 20% SDS 1% b-mercaptoethanol 200 ml 500 ml b-mercaptoethanol Final volume 20 ml 50 ml Filter sterilize
* Do not DEPC treat solutions containing Tris.
* Do not add SDS to solutions containing NA+ because a precipitate will form and impede flow of column
* One can use Tris buffers with SDS in the oligo-dT column, but withoug DEPC treatment. The sodium citrate buffers described above can be treated with DEPC, but no SDS can be used.
From Gene (1983) 25:263-269 via R. Calza