RT in situ PCR for the detection of mRNA transcripts of Fas-L in the immune-privileged placental environment

Abstract

Rapid and reproducible detection of RNA in cells and tissue sections is routinely accomplished using in-situ hybridization technique provided that the target number of mRNA copies is above a minimum number. Detection of low copy transcripts is problematic when threshold detection occurs below clear signal resolution or alternatively, when technical problems result in background noise which occludes clear signal. RT in-situ PCR methodology utilizes both the power and specificity of PCR to amplify target whose localization is subsequently detected at the cellular level. RT in-situ PCR methods routinely involve a two-step methodology. mRNA copies are initially transcribed into cDNA. This step is followed by a separate PCR step wherein amplification of the newly synthesized cDNA takes place. A simplified one-step procedure biochemically compartmentalizes these sequential steps within a single applications methodology using the enzyme rTth. This method was successfully applied to detect and localize mRNA transcripts for Fas ligand within the immune privileged placental environment and to provide verification of immunohistochemical localization of gene product.