Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA.
We have employed an analytical approach for the development of an in situ hybridization methodology using synthetic oligodeoxynucleotide probes for actin messenger RNA detection in cultures of chicken fibroblasts and myoblasts. The methodology developed shows that oligonucleotides can complement the use of nick-translated probes in specific situations. Since they can be made to specific nucleic acid regions independent of restriction enzyme sites, they may be the most convenient approach for analysis of gene families among which sequences are highly conserved. However, it was found that oligonucleotides synthesized to different regions of a messenger RNA behave in situ with differing efficiencies, indicating that not all target sequences are equivalent. Therefore it was necessary to screen several oligonucleotide probes to a target molecule to find the optimal one. The convenience of using synthetic DNA probes makes it worthwhile to explore some of these characteristic properties so as to increase the sensitivity of this approach beyond its application to targets in high abundance.
Department of Anatomy, University of Massachusetts Medical School, Worcester 01605.
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D000199:Actins / Q000096:biosynthesis*; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000032:analysis; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*
D000199:Actins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D004247:DNA