Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction

TLDR

Effective methods are needed to identify and isolate genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The method uses oligonucleotide primers anchored to the polyadenylate tail and arbitrary primers to amplify mRNA subpopulations by reverse transcription and PCR, then resolves the products on a sequencing gel. Using multiple primer sets produced reproducible cDNA fragment patterns that depended strongly on primer sequence specificity.

Abstract

Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.