Microwave Treatment Enhances Detection of RNA and DNA by In Situ Hybridization

Publisher Website
Google Scholar
Abstract
The use of oligonucleotide probes with nonisotopic detection systems has made in situ hybridization (ISH) more accessible for use in diagnostic pathology; however, ISH using formalin-fixed, paraffin-embedded tissues remains much more sensitive when performed with radioactively labeled probes compared with nonisotopic reporter systems, especially using oligonucleotide probes. We investigated the effects of microwave pretreatment on the detection of RNA and DNA in formalin-fixed, paraffin-embedded tissues in an attempt to improve the sensitivity of ISH with digoxigenin-labeled probes. Titration of normal tissues with oligonucleotide probe cocktails for albumin, prolactin, chromogranin (Cg) A and B mRNAs and a cDNA probe for JC virus showed a significant increase in sensitivity with the use of microwave treatment step rather than heating at 70 degrees C in 2 x SSC for 30 min. Analysis of various solutions used for microwaving showed that 10 mM citrate buffer was more effective than 2 x SSC, phosphate-buffered saline, tissue unmasking fluid, or water. A 15- to 20-min period of microwaving in an 800-W oven produced optimum results. Analysis of a group of tumors for albumin and CgA and B mRNAs and infected brain biopsies for JC virus DNA showed increased sensitivity of the microwave technique using digoxigenin-labeled probes. These results show that microwave treatment can enhance the detection of mRNA and DNA in formalin-fixed, paraffin-embedded tissues.