total RNA Amplification:

RNA extraction Protocol for Seed Tissue (and leaf tissue, however the pre-extraction with the buffer solution prior to Trizol is not necessary for leaves. If you go right to Trizol with seed tissue, your RNA pellet will be almost entirely starch. Note: Of course gloves should be worn throughout this procedure. Also, samples should be on ice and all solutions used should be relatively sterile.)

1. Grind sample in liquid Nitrogen. Keep powder frozen. If your sample is not ground to the consistency of flour, your yields will likely be lowered.

2. Add enough powder to a 2 ml tube to equal roughly 0.2 to 0.5 cc of volume.

(Note: if you use much more powder than this, you will not recover all of the RNA, nor will your RNA be very intact or clean.)

3.Add 0.5 ml Extraction Buffer and vortex. (Note: you will need to let your frozen samples warm up slightly before adding this buffer. Of course, if your samples freeze into a cube, they should be thawed and then vortexed again till homogeneous.)

4.Add 0.5 ml Phenol/Chloroform/Isoamyl alcohol (49:49:2) and vortex till thoroughly suspended.

5.Spin 5 minutes at 14,000 RPM in large Sorvall centrifuge at 4 C.

6.Remove 0.5 ml of upper aqueous phase to a fresh 2 ml tube being careful to avoid the interphase material.

7.Add 1.0 ml Trizol reagent and vortex (your sample at this point will be completely incorporated into the Trizol solution and should be invisible).

8.Add 0.2 ml Chloroform and vortex and the phases will separate just like magic.

9.Spin 14,000 for five minutes at 4 C.

10.Remove 0.75 ml of upper aqueous to a fresh 2 ml tube. (This is not all of the upper phase. However, if you contaminate your sample with the interphase material your RNA will be of lower quality and hard to accurately quantify.)

11.Add 0.5 ml Chloroform and vortex.

12.Spin 14,000 for five minutes at 4 C.

13.Remove 0.6 ml of upper aqueous to a fresh 2 ml tube.

14.Add 1.2 ml of 100% ethanol and 60 ul of 3 M Sodium Acetate.

15.Vortex and incubate at -80 C for > 1 hr.

16.Spin 14,000 for 20 minutes at 4 C.

17.Decant supernatants, and wash pellets with 70% ethanol, and briefly respin. Decant or aspirate most of remaining ethanol. Do not completely dry your RNA as it will then be quite difficult to resuspend in TE.

18.Resuspend RNA pellets in 50 ul of sterile TE by vortexing. Expected yield is 25 to 100 ug.

19. Assay 2 ul in 200 ul TE on the spectrophotometer. This should give readings in the 0.1-0.3 range. To examine degree of intactness of your RNA, load 1 ug of each sample on a standard agarose gel and photograph. You should see the two Ribosomal bands and very little if any smearing forward of the ribosomal bands which would indicate degradation. Note: your A260/A280 ratios should be greater than 1.8 with this procedure. They will likely be 2.0 +/- 0.1.

Extraction Buffer (make up fresh day of use.) 20 mls.

50 mM Tris pH 9.0 1 ml 1 M Tris pH 9.0

200 mM NaCl 2 ml 2 M NaCl

1% Sarcosyl 2 ml 10% Sarcosyl

20mM EDTA 0.8 ml 0.5 M EDTA

5 mM DTT 0.1 ml 1M DTT (freezer aliquots.)

H2O 14.1

Remarks: The protocol comes from Mike Giroux, mgiroux@montana.edu

All experiments using protocol P-AFMX-2: (E-AFMX-2, E-AFMX-3)