Selection of oligonucleotide probes for detection of mRNA isoforms

In this article we discuss strategies for selecting oligonucleotides to target isoform-specific mRNAs, drawing on our experience with isotopically labeled oligonucleotides for ISH of Na,K-ATPase mRNA alpha- and beta-subunit isoforms. Oligonucleotide probes based on one of these isoforms have a high probability of forming nonspecific hybrids with related isoform mRNAs. The design and selection of isoform-specific ISH and how their nucleotide structure influences hybridization are reviewed, as well as basic principles in identifying and evaluating candidate probes. Controls such as Tm analysis and GC content are evaluated. For distinguishing among multiple isoforms of gene families, choose lowest possible homology between isoforms consistent with other factors that influence probe performance.