1. Preparation of solutions

1. DEPC-treated water
2. Homogenaze buffer (0.2 Tris HCl, pH 8.2, 0.2 M NaCl, 2% SDS)

a) 1 M Tris HCl, pH 8.2	20 ml
b) 1 M NaCl	20 ml
c) 10% SDS	20 ml
Add DEPC-water to 100 ml Keep at room temperature

Proteinase K stock solution (10 mM Tris HCl, pH 8.0-8.2, 1 mM CaCl₂)

1 M Tris HCl, pH 8.2	1 ml
CaCl2 2H2O (M.W. 147.0)	14.7 mg
Add DEPC water to 100 ml

2. Extraction of total RNA

1. Preparation of proteinase K solution.
   (1) Solve 5.0 mg of proteinase K in 1.0 ml of proteinase K stock solution.
   　-- Solution A.
   (2) Preincubate the homogenizing buffer and Solution A for 15 min at 45℃.
   (3) Mix 1 ml of Solution A and 9 ml of the homogenizing buffer.
   　-- Solution B.
   
2. Put fixed tissue sections (one to four) in autoclaved 1.5 ml tube, add 200 ul of Solution B per one tube, and homogenate sections by vortex.
3. Incubate the homogenates for 1-2 hour at 45℃.
　Vortex for 10 sec at 0, 15, 30, 45, 60 min

4. Add 500 ul of Trizol (Lifetech) into each tube, vortex and leave for 10 min at room temperature.
5. Add 200 ul of chloroform into each tube.
6. Vortex and leave for 10 min at room temperature
7. Spin the tube at 14,000 rpm x 15 min and collect the supernatant (about 500 ul).
8. Add the same volume of isopropanol and vortex.
9. Leave for 10 min at 4℃ to precipitate RNA
10. Spin the tube at 14,000 rpm x 15 min and discard the supernatant
11. Add 500 ul of 70% ethanol and spin the tube at 14,000 rpm x 5 min
12. Discard the supernatant and the pellet is air-dried (30 min, room temperature).
13. Solve the pellet in 10 - 30 ul of DEPC-treated water or TE.
14. Evaluatie the content of RNA with spectrophotometry.