title: An assessment of the efficacy of in situ hybridization as a quantitative method by variance components estimation.
abstract: One serviceable feature of in situ hybridization is its potential for assessing relative levels of mRNA in specific regions of tissues and organs. To determine its efficacy as a quantitative technique, we applied a nested factorial design to a multifactorial experiment. Estimates of the magnitude of variance components then allowed an assessment of variation over samples of sections from the same tissue source, variation in label over 2 anatomical sites within the same section of tissue, as well as experiment-to-experiment variation. We found approximately 51% of the total variance arose from experiment-to-experiment variation, while approximately 21% of the total variance was due to variation in autoradiography grain density over neurons in the same brain region. Rat-to-rat variation accounted for approximately 11%. About 10% of the variance was due to variation between sections of tissue that were derived from the same tissue source and were hybridized in the same hybridization experiment. Variation between 2 homologous, bilaterally located brain regions located on the same tissue section (the right and left supraoptic nucleus), accounted for approximately 5% of the total variance. The remaining unaccounted error variance was approximately 2% of the total variance. Since an expected change in cellular content of a particular mRNA was observed as a function of experimental treatment, results suggest in situ hybridization is a useful quantitative method.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Anatomy & Cell Biology, F.E. Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors.forename: J T
authors.initials: JT
authors.lastname: McCabe
authors.forename: T C
authors.initials: TC
authors.lastname: Kao
authors.forename: M L
authors.initials: ML
authors.lastname: Volkov
mesh_terms: D000818:Animals; D001345:Autoradiography; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D009994:Osmolar Concentration; D010121:Oxytocin / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D013223:Statistics as Topic; D013495:Supraoptic Nucleus / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D010121:Oxytocin