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

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Abstract
We have developed a novel vector-free method for the synthesis of non-isotopic (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.