TY - JOUR
T1 - Quantitative analysis of in situ hybridization methods for the detection of actin gene expression
AU - Bentley Lawrence, Jeanne
AU - Singer, Robert H.
PY - 1985/3/11
Y1 - 1985/3/11
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/13.5.1777
DO - 10.1093/nar/13.5.1777
M3 - Article
C2 - 3889842
AN - SCOPUS:0022423223
SN - 0305-1048
VL - 13
SP - 1777
EP - 1799
JO - Nucleic acids research
JF - Nucleic acids research
IS - 5
ER -