Identification and Cloning of Differentially Expressed Genes by DDRT-PCR

Abstract

The invention of polymerase chain reaction (PCR) and its application to amplification of reverse transcribed cDNA copies of mRNA has opened new possibilities for the development of techniques for identification of changes in gene expression patterns. Two very similar variants of the same strategy were published in 1992 that were named mRNA differential display (1) and RNA arbitrary primed PCR (2). In contrast to the standard RT-PCR technique, where individual mRNAs are detected in a qualitative and sometimes also quantitative manner using gene-specific primers, in these new techniques a set of short primers with arbitrary sequences is used to generate amplified cDNA fragments from a large number of mRNAs used before for DNA fingerprinting (3). Basically, the identification of every individual mRNA species expressed should be possible provided a sufficiently large number of primer pairs is used (4). The method of differential display (DDRT-PCR) has already been described in detail in a number of reviews (5–10). Therefore, we give only a very brief description of the principle. First, RNA is prepared from the different cell types of interest. Second, RNA is reverse transcribed in 12 (1,4), 4 (11), or 3 (12) fractions using anchored oligo-dT primers (e.g., T₁₁VV in Fig. 1) to generate single-stranded cDNA. Third, every cDNA fraction is amplified in a number of independent PCR reactions using various 10-mer upstream primers. Fourth, products of individual PCR reactions are separated on standard sequencing gels (1,11) or on nondenaturing polyacrylamide gels (4,6,10) and visualized, normally by autoradiography. Fifth, fragments differing in their intensities between the parallel samples from different cells are cut out of the gel, reamplified, cloned into a suitable vector, and sequenced. Sixth, differential expression of the genes corresponding to the identified sequence tags is confirmed by an independent analysis. We have previously recommended nuclear run-on analysis (6,10). However, this method is not easy to apply to small amounts of cells and tissues. Therefore, we have recently optimized conditions for RNase protection analysis that we will describe here. Finally, if cloning of full-length cDNAs is the ultimate goal, simultaneous isolation of a larger number of cDNA clones from a suitable library can be carried out using a biotinylated fragment mix as probes and streptavidin-coated magnetic beads to purify specifically hybridizing cDNA clones. The cloning step is not an essential part of DDRT-PCR and it is usually omitted if only diagnosis of gene expression patterns is required.