A method for identifying cDNA clones that hybridize to differentially expressed RNAs is described. Briefly, the RNA population in which the RNAs of interest are more abundant is used as a template for the synthesis of 35S-labeled cDNAs and another RNA population in which the RNAs of interest are less abundant is used as a template for the synthesis of 32P-labeled cDNAs. The labeled cDNAs are pooled and hybridized to plaque or colony lifts constructed from a cDNA library. Clones that hybridize to RNAs that are differentially expressed are identified using differential autoradiography/fluorography to discriminate between the 32P and 35S isotopes. We have used this method to identify cDNA clones that hybridize to mRNAs that are more abundant in the flowers of wild-type tomato than in the flowers of mutants that have low endogenous levels of gibberellins.
Bibliographical noteFunding Information:
We thank J. Berman for making suggestionst hat improved the manuscript. This work was supported by research funds from the Graduate School of the University of Minnesota and a grant from Hoechst AG to Massachusetts General Hospital, R.T.G. is a
Copyright 2014 Elsevier B.V., All rights reserved.
- Lycopersicon esculentum
- Recombinant DNA
- colony hybridization
- differential autoradiography/fluorography
- flower development
- gib-1 mutant
- molecular cloning
- plaque hybridization