In situ RT-PCR Protocol

10X RT Buffer

10X PCR Buffer

100 mM Tris pH 9.0

500 mM KCl

1% Triton X-100

25 mM MgCl ₂

use at a concentration of 1.5 mM

Lysis Solution

4M GuSCN 250 g guanidine thiocyanate

25mM Na citrate 7.0 17.6 ml 0.75M Na citrate pH 7.0

0.5% Sarkosyl 26.4 ml 10% Sarkosyl

add 293 ml Q

before use, add 72ml bME

Water Saturate Phenol

thaw 500 ml phenol

add 0.5 g hydroxyquinolin

add 500 ml Q

mix and allow phases to separate at room temperature

repeat 2 times and store at 4°C

3M NaOAc 5.2

24.6 g NaOAc (anhydrous)

pH to 5.2 with acetic acid

up to 100 ml Q

 

Procedure

rna isolation

• Add tissue to 400 microliters lysis solution with bME added just prior to use. If collecting several samples hold tubes on ice. These can be stored for years at -80°C.

• Add 1 microliter glycogen, 30 microliters 3M NaOAc 5.2 and 500 microliters water saturated phenol. Mix by gentle inversion and add 100 microliters chloroform.

 

• Mix by inversion and hold on ice for 15 minutes.

 

• Spin for 10 minutes at 4°C and remove the aqueous phase. Add 500 microliters isopropanol, hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.

 

• Resuspend the pellet in 300 ml Lysis Solution and add 300 ml isopropanol. Hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.

 

• Resuspend the pellet in 10 ml DEPC treated Q and store at -80°C.