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AU-Differential Display 225
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AU-Differential Display, Reproducibility
of a Differential mRNA Display Targeted to AU Motifs
Orlando Dominguez, Lidia Sabater, Yaqoub Ashhab,
Eva Belloso, and Ricardo Pujol-Borrell
1. Introduction
AU-rich elements (AREs) are found in 3′ untranslated regions (3′ UTR) of many
highly unstable mRNAs for mammalian early-response genes. The minimal AU
sequence core within the ARE is the heptamer WAUUUAW, although from a functional
point of view, several pentanucleotides clustered in close proximity are the key
sequence motif that mediates mRNA degradation (1).
Genes containing AREs are of potential biological and pharmacological interest
because they often code for inflammatory mediators, cytokines, proto-oncoproteins,
and transcription factors (2–5).
A targeted differential display named AU-motif directed display (AU-DD) is
described here. It allows the isolation of cDNA fragments from ARE-containing
mRNAs with minimal sequence information. AU-DD combines a high specificity
gained at the polymerase chain reaction (PCR) level with the advantages of direct
comparison between samples at different stages of activation to detect differentially
expressed genes (6,7).
Previous examples of targeted differential display have used longer and wellconserved
motifs from multigene families. These include zinc finger motifs (8,9),
plant MADS boxes (10), and motifs specific for heat shock proteins (11). They employ
longer conserved sequences (from 6 to 8 codons) to design primers that can predictably
work well on PCR.
The PCR step of AU-DD uses primers which contain two distinctive domains, a
double AU motif at the 3′ end (AU2 in short) and a guanosine rich 5′ (Table 1). This
primer core sequence is previously incorporated as a 5′ tag in the retrotranscription
primer (see below). The 5′ domain configures a sticky anchor that increases Tm and
stabilizes primer annealing whereas the 3′ confers motif specificity. Both 5′ and 3′
domains contribute in a cooperative way to primer annealing. When the natural primerbinding
site was identified on cloned products, it was always found to be a true AU-rich
site. The use of the AU2 domain-based primers does not restrict; however, the spectrum
of amplified mRNAs to clustered AU-pentamer containing genes. In fact, cDNAs
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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