This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

This method is used to detect genomic DNA deletions in tumor cells. For a more detailed discussion of applying this approach to microdissected samples, see Allelic Loss Studies in Prostate MP at NCI.

1. Reagents

DNA sample (see Processing of Microdissected Tissue - DNA-based Analysis)

Proteinase K (Sigma)

Proteinase K buffer (0.05 M tris-HCL, 0.001 M EDTA, 1% Tween 20, 0.1 mg/ml proteinase K, pH 8.0)

Ampli Taq Gold Buffer (Perkin Elmer)

dNTP mixture (Perkin Elmer)

Primers

DEPC-treated H2O

Ampli Taq Gold Polymerase (Perkin Elmer)

a-32P dCTP, 6000 Ci/mmol (NEN Dupont)

Formamide, 99% (Fluka)

Bromophenol blue-Xylene cyanole (Sigma), reconstituted as directed

Gel Mix-6 sequencing gel solution (Life Technologies)

Ammonium persulfate (Biorad)

10X TBE buffer (0.89 M Tris Base, 0.89 M Boric Acid 0.02M Disodium EDTA ) (Advanced Biotechnologies)

Acrylease (Stratagene)

Glass cleaner (e.g., Windex, Glass Plus)

95% ethanol

2. Equipment

Thermal cycler (MJ Research)

Sequencing gel electrophoresis apparatus (Gibco BRL)

High voltage power supply

Gel dryer (Life Technologies)

Glass plates, 31.0 x 38.5 cm (Life Technologies)

0.4 mm spacers (Life Technologies)

Casting boot (Life Technologies)

Shark tooth comb (Life Technologies)

Small clamps

Whatman blotting paper, 3 mm thickness

Kodak Biomax MR or AR film

Film cassette (Amersham Life Science)

Film processor

3. Time Requirements

Gel preparation: 1.5 - 2 hours. Polymerization requires 1 hour, but may stand overnight.

LOH reactions: 2 .5 hours (approximately 1 hour for set-up, 1.5 for PCR)

High-resolution denaturing polyacrylamide gel electrophoresis: 1-3 hours. Twenty minutes for set-up. Electrophoresis time varies according to product size.

Gel drying: 1 hour

Autoradiography: 1 hour-2 days

4. Methods

TIP: Investigators must be especially careful when using this methodology to analyze archival tissue specimens. Formalin fixation in particular results in DNA that is difficult to amplify and often produces inconsistent PCR results, including artifactual allelic loss and poor amplification of large products. Therefore, when this technique is used to analyze archival samples, it is highly recommended that replicate experiments (multiple independent dissections, triplicate PCR reactions, etc.) be used to verify results.

A: LCM and Proteinase K Treatment

Obtain microdissected cells using the LCM procedure.

TIP: The number of cells needed to successfully perform the assay varies depending on the quality and processing conditions of the tissue samples. One thousand cells is recommended as a good starting point.

Suspend approximately 1000 microdissected cells in 20 µl proteinase K buffer.

Incubate overnight at 37°C.

B: Prepare the Glass Plates

TIP: Use Accuwipes for cleaning purposes, as they will not leave lint behind and are non-abrasive.

Clean glass plates twice with glass cleaner.

Repeat using 95% EtOH.

Spray small plate with Acrylease.

Spread Acrylease evenly using a circular motion.

Buff dry.

Quickly assemble the plates without touching the clean surface.

Place 0.4 mm spacers on the edges of the larger plate.

Place the smaller glass plate on top of the larger plate and spacers.

Secure the plates with a casting boot (tape or clamps may be substituted for the casting boot).