Many different methods have been tried in an effort to extract quality RNA from mature conifer needles and phloem, to no avail. Finally here is a method that worked for me. The procedure is adapted from Hughes and Galau, 1988, Plant Molecular Biology Reporter 6(4): 253-257.

The key to the success of this protocol rests, I think, with the initial, high molarity potassium acetate ppt. The RNA is rescued from the bad things that ppt out. Also important are the three detergents (cationic, anionic, and neutral) in the extraction buffer. Don't mess with these two steps--modify other steps if you like. In our hands we found elimination of steps or short cuts after the high molarity ppt still gave RNA , but less pure.

Needle tissue is composed of a large amount of non-living cell wall tissue and a low amount of protoplasm per gram fresh weight. Yields are much lower than with seedling tissue. Seedling tissues can be processed by guanidinium/borate process. I have another protocol for the latter.

Preparation:

Bake glassware and measuring spatulae; treat plastic ware (if not new and sterile) and miracloth with 0.1% diethyl pyrocarbonate (DEP) solution for at least 30 min. DEP-treat water, autoclave and use for making up solutions. Wear plastic gloves and use flamed forcepts to touch lips of tubes or pick up miracloth, etc. Autoclave sheets of filter or blotting paper (to drain tubes).

1. Extraction buffer, 100 ml:

200 mM tris 2.42 g

300 mM LiCl 1.27 g

10 mM Na₄ EDTA 0.38 g

Add 80 ml room temp. H₂ O to above and pH with HCl to pH 8.5 (test with pH strips, not with pH meter to avoid introducing RNA ses).

Then add:

  1.5% w/v N-lauroylsarcosine (Na salt)   1.50 g



     1.0% NP-40 (nonidet P-40)               1 ml



     1.0% sodium deoxycholic acid            1.00 g

Add water to 100 ml, add 100 ul DEP and shake, let stand 30 min then autoclave. (Maybe the DEP doesn't do any good because of the Tris, but I always DEP treat anyway.)

When cool add

     5 mM thiourea                 0.038 g



     1 mM aurintricarboxylic acid  0.0422g



     10 mM dithiotreitol           0.1542g

2. 8.5 M potassium acetate, pH 6-6.5: 83.4 g K acetate + 20 ml H₂ O with 11.5 ml glacial acetic acid, stir, autoclave--goes into solution with autoclaving...test pH by diluting an aliquot of stock solution--accurate pH cannot be determined from the concentrated solution. Adjust volume if necessary. Upon cooling the solution becomes a glue-like mass. Measure amounts by weight.

3. 3.3 M sodium acetate, pH 6.1: 27.07 g + 60 ml H₂ O adjust pH with acetic acid and adjust volume to 100 ml. Autoclave.

4. 10 M LiCl; 21.2 g to 50 ml with H₂ O. Autoclave.

5. 5 M potassium acetate: 49 g to 100 ml with H₂ O. Autoclave.

6. TE: 10 mM tris-HCl with 1 mM EDTA ph 7.6.

7. isopropanol: cold

8. ethanol: cold

9. chloroform/butanol: 4:l

The following is protocol for 1.6 g of needle or phloem tissue, which is a suitable volume for one 50-ml orange cap tube (Corning, conical) that you will need for processing. Scale up by nX the following protocol:

Chill extraction buffer. Grind tissue with dry ice in coffee grinder. Transfer powder to a sterile, new 50-ml polypropylene orange cap tube with volume marks. When dry ice has evaporated but tissue is still frozen add 20 ml extraction buffer. Cap and vortex vigorously, 30 sec. Or use dep-soaked polytron homogenizer and blend on low-medium for 30 sec (can increase yields by 1.5x). Place on dry ice or in -80 freezer to freeze quickly. Partially thaw in 37℃ water bath. Vortex vigorously and continue thawing. Vortex vigorously when thawed but still cold. Put on ice, add 1/3 weight/volume 8.5 M potassium acetate, pH 6-6.5; mix by inversion 10X and incubate on ice 15 min. Centrifuge on high in clinical centrifuge in cold for 20 min. Transfer the yellow-green, clear supernatant through one layer of miracloth into a new 50 ml orange cap tube. Add 1/9 volume of 3.3 M sodium acetate, pH 6.1 (I think you really don't have to do this, but I always do anyway before the isopropanol ppt) and 0.5 X (of the second volume) cold isopropanol. Mix and store at -20 for 2 hrs.

Centrifuge the isopropanol solution on high in clinical centrifuge in the cold for 20 min. Discard supernatant and drain tubes on sterile paper. Dry under vacuum 5 min. Phloem extraction gives a large, pinkish, gelatinous pellet; needle extraction gives a small greenish pellet. Resuspend the phloem pellet in 3 ml of TE; resuspend the needle pellet in 1.6 ml TE. Heat at 65℃ for 5 min; pipet the solution up and down with pipetman. Centrifuge on high in clinical centrifuge 5 min. Remove supernatant, measure volume, and add 1/4 vol 10 M LiCl to make solution 2 M and incubate in refrigerator overnight. Centrifuge the LiCl solution at 10,000 g for 30 min in the cold. Discard the supernatant and dissolve the pellet in 400 ul TE. Allow 15 min at room temp. for complete dissolution with frequent mixing. Spin 2 min. in microfuge at room temp. Remove supernatant. Discard pellet.

Here you have a choice: You can layer the RNA /TE over CsCl step gradient or proceed with purification that does not require 24 hr of centrifugation. Here is the non-centrifugation procedure:

To supernatant add 1.5 volume 5 M potassium acetate (pH NOT adjusted) mix and incubate on ice for 3-5 hr. Centrifuge at 10,000g for 30 min in the cold. Discard the supernatant. Resuspend the white pellet in 400 ul TE. Extract with an equal volume of chloroform/butanol (4:1); pipette off supernatant and save, and re-extract the chloroform/butanol with TE two more times, pooling supernatants. Spin pooled supernatants at room temp 2 min to remove contaminants. Transfer supernatant in 400 ul aliquots to eppendorfs or transfer all to silanized corex tube--if you scaled up the procedure to more tubes. Add 1/9 vol of 3.3 M sodium acetate pH 6.l and 2 volumes (of the second volume) cold ethanol. Mix and place at -20 for at least 2 hrs to overnight. Spin 9,500 g for 30 min in the cold, drain. Resuspend pellet in 80% ethanol, spin at room temp 5 min (high speed in microfuge), carefully drain and aspirate liquid. Partially dry the pellet. Dissolve in 60ul water (or more depending on scale-up) . Use 5-10% of the sample to determine the spectrum (dilute with water). Store sample(s) at -80℃. Mature needle RNA seems to degrade easily. Therefore, after I determine concentration of the sample I aliquot RNA into portions for storing to avoid freezing and thawing the stock.

Yield: using vortexing for extraction, yield was 56.3 ug total RNA /g tissue. In a scale-up using the polytron homogenizer, yield was 83 ug total RNA /g tissue.