116 Internaiional zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR Journal of Cardiology, 30 (1991) 116-118 Elsevier CARD10 11822 Detection of myosin gene expression in the developing heart using probes derived by polymerase chain reaction Paul J.R. Barton ‘, Gonzalo Moscoso ’ and Robert P. Thompson 3* ’ Department of Cardiothoracic Surgery and 3 Department of Paediatrics, National Heart and Lung Institute, London and ’ Department of Morbid Anatomy, Kings College Hospital Medical School, London, U.K. (Received 28 June 1990; revision accepted 6 July 1990) zyxwvutsrqponmlkjihgfedcbaZYXWVU The polymerase chain reaction provides a rapid method for the molecular cloning of DNA probes suitable for the detection of specific messenger RNA. We have used this approach to prepare probes zyxwvutsrqponmlkjihgfedcbaZYXWV specific for human cardiac my& messenger RNA and demonstrate here the use of such probes in the analysis of human cardiac development by hybridization in situ to sections of fetal tissue. This combination of kchnfques is suitable for the detection of any messenger RNA for which sequence data are available, and offers a powerful new approach to the analysis of cardiac development. Key words: Myosin; Hybridization; Gene expression; Cardiac development Introduction The analysis of gene expression is of fundamental importance to our understanding of the molecular mechanisms underlying cardiac development. Provided a suitable cloned probe is available, the accumulation of messenger RNA which occurs during gene expression can be readily assayed by a variety of methods based on nucleic acid hybridization. Sequence data for a large number of human genes are now available [l] and the advent of the polymerase chain reaction (generally re- ferred to as PCR) as a rapid and convenient method for molecular cloning has greatly simplified the process of preparing specific hybridization probes [e.g. 2,3]. These techniques, together with the ability to detect the accu- mulation of messenger RNA in situ by the direct hy- Correspondence to: P.J.R. Barton, Dept. of Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, U.K. * Visiting Fellow from the Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston SC. USA. bridization of radioactively labeled DNA or RNA probes on tissue sections [4], provide the necessary tools for analysing gene expression during human cardiac development. We demonstrate this approach here by the analysis of myosin gene expression in a 12 week human fetal heart. Materials aud Methods Using techniques based on the polymerase chain reaction we have cloned 104 nucleotides of human /3-myosin heavy chain 3’ non-coding sequence (clone pHMB-1 [2]). The preparation of radioactive hybridiza- tion probe from this clone, preparation of tissue, as well as in situ hybridization, were carried out essentially as described [4]. Briefly, human fetal material was ob- tained from therapeutic abortions and fixed by perfu- sion with paraformaldehyde prior to mounting in paraf- fin and sectioning (7 microns). The 35S-labelled probe was produced by transcription from the cloned frag- ment and hybridized overnight to prepared sections. Following removal of unhybridized probe and autoradi- ography, sections were examined for the presence of silver grams in the photographic emulsion under dark- field illumination. 0167-5273/91/$03.50 0 1991 Elsevier Science Publishers B.V. (Biomedical Division)