A simple method of in-situ hybridization (ISH) without using any bacteriological or recombinant procedures was exemplified by examining the localization of the two isoforms of calcineurin catalytic subunits, Aα and Aβ, in rat brain tissue. Total RNA was extracted from rat brain tissue and utilized as a template to perform probe labeling during reverse transcription and subsequent polymerase chain reaction steps (RT-PCR) by incorporating digoxigenin-11-dUTP. These PCR probes were employed for ISH. To confirm the validity of this method, the expression of mRNAs of alpha-fetoprotein and albumin in rat liver tissue was also examined using the same procedure. The distribution of calcineurin Aα and Aβ mRNAs was clearly detected in brain tissue. The entire procedure took less than a few days from starting the preparation of the probes to the light microscopic observation of the specimens. Moreover, the sensitivity of the ISH using these probes was higher than 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.
- Alpha-fetoprotein (AFP)
- DNA probe
- In-situ hybridization
- Polymerase chain reaction (PCR)
ASJC Scopus subject areas
- Pathology and Forensic Medicine
- Cell Biology