How To Find and Clone the Appropriate cDNA Fragments Generated in Differential mRNA Display by Using Northern Blot for cDNA Capture

Abstract

A new technique called differential mRNA display has been recently established and used to identify differences in subsets of mRNA samples (Liang and Pardee 1992; Liang et al. 1992). This technique enables one to analyze any mechanism which involves changes in specific mRNA levels. By utilizing this technique, differentially expressed genes with known or previously unreported sequences have already been identified (Sager et al. 1993; Aiello et al. 1994; Zou et al. 1994). The key element of this technique is the use of sets of anchored and arbitrary primers to generate cDNA fragments in reverse transcription followed by polymerase chain reactions (RT-PCRs). The cDNA fragments are resolved and compared on sequencing gels. The resulting cDNA patterns reflect differences in the mRNA levels and composition. Differentially displayed cDNAs are isolated, cloned, sequenced and used as probes on northern blots to confirm differences in the particular mRNA level. The general strategy seems to be simple and straightforward, although increasing number of reports on methodical refinements (Liang et al. 1993; Li et al. 1994; Reeves et al. 1995) suggest that the involved procedures are technically very challenging.