Gerard J. Nuovo

Director, MGN Medical Research Laboratories, Setauket, New York 11733, USA

Received: 07/25/96; Accepted: 10/02/96; On-line 11/01/96

4. THE ONE STEP RT in situ PCR PROTOCOL

This next section will detail the protocols used for detection of PCR amplified cDNA and DNA.

RT in situ PCR (17)

1) Place three 4µ tissue sections or three cells suspensions on a silane coated slide (ONCOR, Gaithersburg, MD).

2) Remove paraffin (tissue sections) with 5 min xylene wash, 5 min 100% ethanol wash.

3) Digest with pepsin (2 mg/ml) for optimal time (usually 30-90 min).

4) Incubate two of three sections with 10-20U DNase overnight at 37°C.

5) Wash for 1 min with DEPC water, then 100% ETOH, and air dry.

6) Prepare the following solution from the EZ rTth kit (Perkin Elmer):

10 µl EZ buffer(EZ rTth RNA PCR kit)

1.6 µl each of dATP, dCTP, dGTP, dTTP(each 10 mM stock)

1.6 µl of 2% bovine serum albumin

1.0 µl of Rnasin (2 U/(l)

3.0 µl of primers 1 and 2 (20 µM stock)¹

0.6 µl digoxigenin dUTP (1 mM stock)

14.6 µl DEPC water

12.4 µl 10 mM MnCl2

2.0 µl rTth (2.5 U/(l)

1 for the negative control, use nonspecific primers or omit the primers

7) Apply 10-40 µl of above solution to each section, cover with amplicover (Perkin Elmer 1000 cycler) or polypropylene coverslip and anchor with nail polish and mineral oil overlay

8) Incubate at 65°C for 30 min

9) Denature at 94°C for 3 min

10) Cycle at 60°C for 1.5 min, 94°C for 45 sec; do 20 cycles

11) Wash slides in 0.1XSSC/0.2% BSA at 60°C for 15 min

12) Detect digoxigenin as per protocol of Boehringer Mannheim, which follows:

Antidigoxigenin-alkaline phosphatase conjugate at 1:150 in 0.1M TRIS pH 7.5 and 0.1M NaCl for 30 min at 37°C, followed by 10-15 min at 37°C in NBT/BCIP solution = 50 µl of substrate 1 and 50 µl of substrate 2 (Digene Diagnostics, Gaithersburg, MD) in 15 ml of 0.1M TRIS, pH 9.5 and 0.1M NaCl) counterstain for 1-5 min in nuclear fast red.