( centchanges in the conditions used d...)Identification of Differentially Expressed Gene Products with the CastAway,,,System*

cent changes in the conditions used during DD have made DD closer in nature to RAP-PCR.³ The PCR products generated in RAP-PCR and DD are subjected to gel analysis using high-resolution polyacrylamide gel electrophoresis (PAGE). Bands of interest are excised from the gel, reamplified, cloned and sequenced. Differential expression of the cloned gene product is verified using Northern or reverse Northern blotting.⁴

Because of the versatility of these approaches for identifying differentially expressed genes, the most difficult and time-consuming portion of the techniques is the ultimate characterization of the gene product. In this article, we examine the utility of Stratagenes new CastAway prepoured sequencing gels for analyzing PCR products. Using CastAway gels eliminates tedious pouring of polyacrylamide sequencing gels and makes analysis of RAP-PCR or DD faster and easier. The thin, 0.25-mm CastAway gels can be used to quickly run samples, identify differential gene products of interest and isolate and purify band fragments from the gel.

Methods

figure 1

The CastAway precast system and sequencing gels were used for analysis of several RAP-PCR products. The system under investigation consisted of a human myelomonocytic cell line (HL60) that was stimulated for 4 hours with the tumor promoter agent, phorbol myristate acetate (PMA) at a final concentration of 16 ng/ml. We sought to detect immediate-early genes whose expression was modulated in response to this treatment. Messenger RNA was isolated from the control (nonstimulated) cells and the PMA-stimulated cells and was used in RAP-PCR according to the protocol provided with Stratagenes RAP-PCR Kit. Three different arbitrary primers were used for three separate
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