We have developed a novel vector-free method for the synthesis of noni
sotopic (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 promo
ters were incorporated into the amplified DNA by including the promote
r sequences in the 5' end of the oligonucleotides used to prime the PC
R. These promoters allowed the subsequent transcription of digoxigenin
labeled antisense and sense cRNA probes from the amplified DNA. We su
ccessfully 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 s
ingle cell level. This approach avoided time consuming steps which req
uired subcloning of target DNA into the vectors that contains bacterio
phage RNA polymerase promoter as well as the need for radioactive mate
rials. This non-isotopic ISH procedure takes less than 72 h from speci
men preparation to microscopic analysis and should prove to be useful
for molecular biological studies of hormones and clinical diagnosis.