Advantages of in situ hybridisation over direct or indirect in situ reverse transcriptase-polymerase chain reaction for localisation of galanin MRNA expression in rat small intestine and pituitary

In situ hybridisation (ISH) and direct or indirect in situ reverse transcri ptase-polymerase chain reaction (RT-PCR) were used to detect galanin mRNA i n paraffin sections of rat intestine and pituitary. With conventional ISH, a subset of intestinal neuronal ganglion cells and anterior pituitary endoc rine cells were labelled. Direct in situ RT-PCR also labelled some cells in pituitary but not in intestine. Negative controls were unlabelled, but sec tions with 3' primer alone for RT-PCR appeared positive. No signal was appa rent using the indirect in situ RT-PCR method. Investigation of the specifi city of solution phase RT-PCR using RNA extracts from pituitary or intestin e revealed that additional PCR products were detected under some conditions . The sequences of these PCR products suggested that one was the result of mispriming and single primer PCR, which could also have occurred in situ. A lternative galanin primers gave only the predicted RT-PCR product in soluti on phase yet still gave artefacts in tissue sections using direct in situ R T-PCR. ISH with probes transcribed from the correct PCR product gave identi cal labelling to the original galanin riboprobe. In conclusion, direct in s itu RT-PCR is unreliable and requires validation, while indirect in situ RT -PCR may fail even though sufficient target exists for detection with conve ntional sensitive riboprobe ISH.