Target Discovery and Validation Reviews and Protocols

120 Røsok and Sioud
1.1.7. Differential Display (DD)
In message display techniques, PCR-amplified fragments representing the
transcripts are visualized on a gel, revealing the differences in gene expression
between populations of two or more sources. No subtraction is included.
Differential display was developed as a quicker alternative to subtractive
hybridization techniques to obtain cDNAs representing differentially expressed
transcripts (22). The basic principle of the technique is to isolate total RNA or
mRNA from the cell types to be compared and then to reverse transcribe the
RNA by using three different “anchor primers” containing a poly(dT) region
that targets the polyA region of eukaryotic mRNA. The primers differ from
each other in the last 3′ non-T base, fixing the priming site at the beginning at
the polyA tail of three different RNA populations and limiting the number of
RNA species transcribed in each reaction. PCR amplification is performed
using the anchor primers in combination with a short arbitrary primer. The
primers originally used for DD were 14-mer anchor primers with two- (22) and
then one-base–anchored primers (23) to ensure specific priming in the 3′ end.
Since then, the selection of primers used for DD has evolved (24). A related
method known as RNA arbitrarily primed PCR (RAP-PCR) that uses only
primers of random sequence was developed in 1992 (25). The objective of both
strategies is to amplify fragments of sufficient length to allow unique identification
of the mRNA species they represent. As a rule, fragments from 150 to
500 bp can be resolved by size on a DNA sequencing gel (22). PCR primers and
reaction conditions must be chosen to such that a sufficiently small number of
fragments, typically 50–100 fragments are amplified. This amplification can be
achieved by initially running PCR at low stringency to generate mismatching at
a controlled frequency (24). To obtain a comprehensive representation of the
expressed genes of a given cell type, many combinations of anchor and arbitrary
primer must be used. The amplified cDNAs from the different cell types
are separated by size in adjacent lanes of sequencing gels for comparison. To
identify putative differentially expressed genes, the amplified fragments must
be visualized with either isotopes or fluorescent dyes. Putative differentially
expressed fragments can be excised and eluted from the dried gel and then
reamplified.
Different strategies for analyzing the candidate fragments have been
reported. All strategies involve cloning, sequencing, identification, and confirmation
of differential expression; however, these steps occur in a variety of
orders. Some approaches streamline the process by incorporating direct
sequencing of DD fragments (26). However, this strategy is limited to fragments
generated by different primers on each end. Database matching ultimately
identifies readable sequences.