Protocol R.4

RT-PCR Analysis

Solutions

10X RT Buffer

10X PCR Buffer

100 mM Tris pH 9.0

500 mM KCl

1% Triton X-100

25 mM MgCl2

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.

 

reverse transcription reaction

• Add 5 microliters RNA to a sterile siliconized eppendorf tube along with 1 microliter of oligo dT (0.25 mg/ml).

• Heat to 65° for four minutes and immediately transfer to ice.

• Quickly spin the RNA/oligo dT and add 14 microliters of the RT cocktail:

2 microliters 10X RT buffer

1 microliter 1 mg/ml BSA

1 microliter 20 mM DTT

0.5 microliters RNaseIN

0.4 microliters 25 mM dNTPs

0.2 microliters AMV RT

9 microliters Q

• Incubate at 48° for 30 minutes, dilute 5 fold and store at -20°.

pcr reaction

• Dilute cDNA (5 fold) and use between 1-5 microliters of cDNA per PCR reaction.

• Add the following PCR cocktail per tube:

2.5 microliters 10X PCR buffer

1.5 microliters 25 mM MgCl2

1 microliter each primer (10pmol/microliter; 55° Tm)

0.2 microliters 25 mM dNTPs

0.025 microliters a-32P dATP

0.02 microliters Taq polymerase (protocol T.2)

14 microliters Q

• Perform PCR using the following cycle parameters:

94° for 4 minutes, (94° for 30 seconds, 55° for 30 seconds, 72° for 30 seconds) x n, 72° for 5 minutes.

• The number of cycle should be determined empirically to find the linear range of amplification.