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 Na₄EDTA                 0.38 g

Add 80 ml room temp. H₂O to above and pH with HCl to pH 8.5 (test with pH strips, not with pH meter to avoid introducing RNAses).

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 H₂O 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 H₂O adjust pH with acetic acid and adjust volume to 100 ml. Autoclave.

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