Analysis of Gene Transcription In Situ: Methodological Considerations and Application

Abstract

The analysis of gene transcription in situ requires the localization of specific mRNA species in cell or tissue preparations. The main technique used to date is in situ hybridization (ISH) employing labeled nucleic acid probes complementary to the specific mRNA of interest. Advances in recombinant DNA technology and probe labeling methods, including the introduction of non-isotopic labels, have made the technique increasingly accessible. Further refinements in the methodology have enabled the detection of mRNA sequences in a variety of fixed cell and tissue preparations, including processed tissues, both at light and electron microscopy levels. When ISH is combined with immunohistochemistry, valuable insights into the molecular basis of disease pathogenesis in vivo can be gained.

This review concentrates on the methodological considerations for ISH and its applications in pathology. Probe labeling, sample preparation, hybridization, and detection procedures are discussed. Alternative methods of in situ mRNA detection, such as in situ polymerase chain reaction (IS-PCR) and primed labeling in situ (PRINS), are also considered. (The J Histotechnol 17:203, 1994)

Keywords:

• cDNA probes
• in situ hybridization
• in situ PCR
• mRNA
• oligonucleotide probes
• PRINS
• probe labeling
• ssRNA probes