( CastAway precast gels for fast analys...)Identification of Differentially Expressed Gene Products with the CastAway,,,System*

CastAway precast gels for fast analysis of RAP-PCR and differential display

Douglas McKenzie and Douglas Drake
Stratagene Cloning Systems, Inc.

Stratagenes new CastAway precast sequencing gels were used to detect and isolate differentially expressed gene products amplified by the RNA arbitrarily primed polymerase chain reaction (RAP-PCR). A human myelomonocytic cell line (HL60) was stimulated for 4 hours with phorbol myristate acetate (PMA). Arbitrary primers were used for first-stand synthesis and PCR amplification of the cellular mRNA. The PCR products were run on the CastAway system, and the pattern of PCR bands was compared to the pattern from a nonstimulated control. Fourteen unique bands representing differentially expressed gene products due to the PMA stimulation were identified, isolated from the gel and reamplified by PCR for further analysis. Differential expression was confirmed by Northern blot analysis or reverse Northern blotting.

Differential gene expression provides the basis for many fundamental cellular processes associated with differentiation and development. The identification and cloning of genes whose expression is modulated is an important step in advancing our understanding of these phenomena. Recently, two different PCR-based techniques were described that provide both the means and the power for analyzing gene transcription on an extensive scale. These techniques, referred to as RNA arbitrarily primed polymerase chain reaction (RAP-PCR)¹ and differential display (DD),² share several features. Both techniques rely on the degenerate binding of arbitrarily chosen primer(s) to sample multiple cDNA species during the polymerase chain reaction (PCR) and, following adequate amplification, yield a unique fingerprint reflective of these species. Although the two techniques were originally quite distinct, re
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