Rapid nonradioactive in-situ hybridization for mRNA detection of calcineurin in rat brain using PCR DNA probe

A simple method of in-situ hybridization (ISH) without using any bacteriolo gical or recombinant procedures was exemplified by examining the localizati on of the two isoforms of calcineurin catalytic subunits, Act and AB, in ra t brain tissue. Total RNA was extracted from rat brain tissue and utilized as a template to perform probe labeling during reverse transcription and su bsequent polymerase chain reaction steps (RT-PCR) by incorporating digoxige nin-11-dUTP. These PCR probes were employed for ISH. To confirm the validit y of this method, the expression of mRNAs of alpha-fetoprotein and albumin in rat liver tissue was also examined using the same procedure. The distrib ution of calcineurin A alpha and A beta mRNAs was clearly detected in brain tissue. The entire procedure took less than a few days from starting the p reparation of the probes to the light microscopic observation of the specim ens. Moreover, the sensitivity of the ISH using these probes was higher tha n that of the end-labeled probe. This method has significant advantages as it allows laboratories without any recombinant facilities to accomplish ISH , and has the potential for widespread application.