( first-stand cDNA synthesis and further...)Identification of Differentially Expressed Gene Products with the CastAway,,,System*

first-stand cDNA synthesis and further PCR amplification reactions in the presence of [a-³²P]dATP. Following PCR amplification, an aliquot of the RAP-PCR products obtained with each cell population was resolved for 2 hours at 50 watts on a CastAway prepoured 6% polyacrylamide gel. The gel was fixed and dried according to the protocol provided with the product. The dried gel was exposed to film overnight and developed. The RAP-PCR fingerprints obtained with each of the arbitrary primers are shown figure 1.

Results

figure 2

We identified 14 different PCR products whose expression was modified as a result of PMA stimulation. The expression of 3 of these products was down regulated in response to PMA, whereas the expression of 11 products was elevated. Primers 1 and 2 yielded differential gene candidates, while none were observed with primer 3. The size of the products ranged from 100 to 550 bp. The gel was reexposed to a second film, and candidate bands were outlined with ink and given an identifying number on the second film figure 2. The dried gel was rehydrated in water overnight and then placed on the second film. Glogos II autorad markers that had been affixed to the glass plate prior to the second exposure provided the means for aligning the bands marked on the film with the corresponding region of the rehydrated gel. A pipettor bearing a 1000-l aerosol- resistant tip was used to repeatedly aspirate the gel sections at the positions corresponding to the band of interest. The pipet tip containing the aspirated gel section was placed into a microcentrifuge tube along with 50 l
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