Quantitative analysis of in situ hybridization methods for the detection of actin gene expression

Jeanne Bentley Lawrence, Robert H. Singer

Research output: Contribution to journalArticle

153 Scopus citations

Abstract

We have implemented an efficient, quantitative approach for the optimization of in situ hybridization using double-stranded recombinant DNA probes. The model system studied was actin mRNA expression in chicken embryonic muscle cultures. Actin and control (pBR322) probes were nick-translated with P32 labeled nucleotides, hybridized to cells grown on coverslips, and quantitated in a scintillation counter. Cellular RNA retention was monitored via the incorporation of H3-Uridine into RNA prior to cell fixation. Over a thousand samples were analyzed, and among the technical variables examined were the fixation protocol, proteolytic cell pretreatment, the time course of hybridization, saturation kinetics, hybridization efficiency, and effect of probe size on hybridization and network formation. Results have allowed us to develop a reproducible in situ hybridization methodology which is simpler and less destructive to cellular RNA and morphology than other protocols. Moreover, this technique is highly sensitive and efficient in detection of cellular RNAs. Lastly, the rapid quantitative approach used for this analysis is valuable in itself as a potential alternative to filter or solution hybridizations.

Original languageEnglish (US)
Pages (from-to)1777-1799
Number of pages23
JournalNucleic acids research
Volume13
Issue number5
DOIs
StatePublished - Mar 11 1985
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

Fingerprint Dive into the research topics of 'Quantitative analysis of in situ hybridization methods for the detection of actin gene expression'. Together they form a unique fingerprint.

  • Cite this