AUTOMATED RECORDING OF RNA DIFFERENTIAL DISPLAY PATTERNS FROM PIG GRANULOSA-CELLS

We have developed a protocol for fast, nonradioactive, mRNA differenti al display reverse transcription PCR (DDRT-PCR) based on a commerical automated sequencer with RNA isolated from pig granulosa cells. We sou ght to discover conditions that would minimize the problem of using re latively small primers labeled with large infrared dye molecule, IR41, required for the sequencer Extended IR41-labeled primers IR41-AAGC-T- 11-A, IR41-AAGC-T-11-C and IR41-AAGC-T-11-G gave more consistent diffe rential display patterns than shorter anchored primers (IR41-T(11)A, I R41-T11C and IR41-T(11)G) without the additional (AAGC) cloning site. The optimal concentration of the extended labeled (downstream) primers was 20 pmol when 13-mer arbitrary (upstream) primers were used at a c oncentration of 4 pmol. Background smear and the intensity of amplifie d bands was significantly improved by changing from conventional Tag D NA polymerase to AmpliTaq(R) Gold(TM) polymerase, which permits an imp roved ''hot start'' for the reaction. Running time (during which a dig itized gel image is recorded) for a 26-cm polyacrylamide gel was 4 h, enabling us to analyze 90 reactions in an 8-h day. This protocol offer s a rapid and reliable nonradioactive method for comparing gene expres sion patterns for various research or diagnostic purposes.