A PCR-Derived, non-isotopic labeled prolactin cRNA probe suitable for in situ hybridization

Abstract

We have developed a novel vector-free method for the synthesis of nonisotopic (digoxigenin) labeled prolactin (PRL)-gene specific cRNA probe based on the direct in vitro transcription of DNA template amplified by polymerase chain reaction (PCR). The T7 and T3 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the '5 end of the oligonucleotides used to prime the PCR. These promoters allowed the subsequent transcription of digoxigenin labeled antisense and sense cRNA probes from the amplified DNA. We successfully utilized these probes to detect specific PRL mRNA in human pituitary and decidua tissues by in situ hybridization (ISH) which can provide identification and localization of PRL-gene expression at a single cell level. This approach avoided time consuming steps which required subcloning of target DNA into the vectors that contains bacteriophage RNA polymerase promoter as well as the need for radioactive materials. This non-isotopic ISH procedure takes less than 72 h from specimen preparation to microscopic analysis and should prove to be useful for molecular biological studies of hormones and clinical diagnosis.

A new PCR-based approach for the preparation of RNA probe

“…A few reports [4][5][6] have described the generation of PCR-derived RNA probes for ISH, in which T7 or T3 RNA polymerase promoters are introduced at the 5V end of genespecific oligonucleotide primers. In comparison to this method, our approach is more convenient in that the same primers used for real-time PCR amplification can also be used for the preparation of RNA probes.…”

“…Preparing RNA probes for novel mRNA, for instance, is still a time-consuming work. A few reports [4][5][6] have described the generation of PCR-derived RNA probes for ISH. However, those methods require extra gene-specific oligonucleotide primers carrying T7 or T3 RNA polymerase promoter site at the 5V end of.…”

In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease

“…In this technique, however, the lack of negative control with sense DNA and the adjustment to optimize RNA-DNA rather than DNA-DNA hybridization limit the reliability and sensitivity of the ISH. PCR, widely used in both research and diagnostic fields, has also been applied in ISH studies, generation of vector-free probes (Wu et al 1995), and in situ PCR techniques (Nuovo et al 1991). An asymmetric PCR has efficiently generated radiolabeled DNA probes for ISH, and Northern and Southern blotting (Scully et al 1990).…”