Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization.
An unusual feature of the mammalian genome is the number of genes exhibiting monoallelic expression. Recently random monoallelic expression of autosomal genes has been reported for olfactory and Ly-49 NK receptor genes, as well as for Il-2, Il-4 and Pax5. RNA fluorescence in situ hybridization (FISH) has been exploited to monitor allelic expression by visualizing the number of sites of transcription in individual nuclei. However, the sensitivity of this technique is difficult to determine for a given gene. We show that by combining DNA and RNA FISH it is possible to control for the hybridization efficiency and the accessibility and visibility of fluorescent probes within the nucleus.
van Raamsdonk
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CD
Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Tilghman
S M
SM
D000483:Alleles; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics; D005456:Fluorescent Dyes; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D005136:Eye Proteins; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012333:RNA, Messenger; D012097:Repressor Proteins; D004247:DNA