An assessment of the efficacy of in situ hybridization as a quantitative method by variance components estimation
✍ Scribed by Joseph T. McCabe; Tzu-Cheg Kao; Marina L. Volkov
- Publisher
- John Wiley and Sons
- Year
- 1993
- Tongue
- English
- Weight
- 804 KB
- Volume
- 25
- Category
- Article
- ISSN
- 1059-910X
No coin nor oath required. For personal study only.
✦ Synopsis
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 a n 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 -51% of the total variance arose from experiment-to-experiment variation, while -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 -5% of the total variance. The remaining unaccounted error variance was -2% of the total variance. Since a n 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. Findings also indicate, however, the importance of experimental design: the use of multiple samples of tissue from the same tissue source, a sufficient number of tissue sources (animals, batches of cultured cells) to account for variations in sample sources, and the need to assess experiment-to-experiment and rat-to-rat variations. Results suggest the utility of analyzing the data of in situ hybridization experiments from the perspective of experimental design.