Rational design of oligonucleotide probes to avoid optimization steps in in situ hybridization

In situ hybridization (ISH) is a widely used technique in neuroscience since it allows a relatively straightforward determination of gene expression in the brain, in respect to distribution as well as in respect to quantification. It is based upon the hybridization of a nucleic acid probe with the mRNA under investigation and does not require the creation of specific antibodies as in immunohistochemistry. However, a major drawback of ISH is the fact that all standard protocols available include time consuming optimization steps of several critical parameters such as tissue fixation, hybridization conditions and washing procedures. Therefore, the aim of our investigation was a rational design of oligonucleotide probes which were adapted to our standard ISH protocol and which could therefore be used without changing any parameter. This approach also worked well for the detection of rare gene products such as neuropeptide receptor mRNAs. To adapt the probes to our standard procedure, sequence, calculated melting temperature, length and secondary structures of the oligonucleotides were considered according to certain constraints as outlined in the following.