Rapid and Reliable Differential Display from Minute Amounts of Tissue: Mass Cloning and Characterization of Differentially Expressed Genes from Loblolly Pine Embryos

Abstract

Performing RNA differential display analysis on small tissue samples is difficult since much RNA, the initial template for the reaction, is lost during conventional isolation procedures. We have developed a rapid method which employs oligo-dT beads to capture mRNA from cell lysates. Subsequent reactions are primed directly from the beads, thus RT and PCR reactions can be completed within a few hours of tissue harvest. This approach allows us to perform differential display on a single pine embryo. We describe strategies for distinguishing classes of co-migrating bands excised from differential display gels and outline a PCR-based method for confirming differential expression of large numbers of cloned bands in cases where RNA quantities are limiting.