An assessment of the efficacy of in situ hybridization as a quantitative method by variance components estimation.
An assessment of the efficacy of in situ hybridization as a quantitative method by variance components estimation.
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通过方差分量估计评估原位杂交作为定量方法的功效。
DOI：
10.1002/jemt.1070250109
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发表时间：
1993
影响因子：
2.5
通讯作者：
Volkov,ML
中科院分区：
文献类型：
--
作者：
McCabe,JT;Kao,TC;Volkov,ML
原位杂交的一个有用的特征是它有可能评估组织和器官特定区域的相对mRNA水平。为了确定其作为一种定量技术的有效性，我们将嵌套析因设计应用于多因素实验。然后，对变异分量大小的估计允许评估来自相同组织来源的切片样本的变异、同一组织切片内两个解剖部位的标记变异以及实验到实验的变异。我们发现，∼51%的总方差来自于实验之间的差异，而∼21%的总方差是由于同一脑区神经元放射自显影颗粒密度的差异。鼠与鼠之间的差异约占11%。约10%的差异是由于来自相同组织来源并在同一杂交实验中杂交的组织切片之间的差异。位于同一组织切片上的两个同源两侧脑区(左右视上核)之间的变异可解释总变异的∼5%。剩余的未解释误差方差为∼总方差的2%。由于特定mRNA的细胞含量在实验处理过程中观察到了预期的变化，结果表明原位杂交是一种有用的定量方法。然而，调查结果也表明了实验设计的重要性：使用来自同一组织来源的多个组织样本，足够数量的组织来源(动物、培养细胞批次)以考虑样本来源的差异，以及评估实验到实验和大鼠到大鼠的差异的必要性。结果表明，从实验设计的角度分析原位杂交实验的数据是有用的。©1993 Wiley-Liss，Inc.
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 ∼ 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 variationbetweensections 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 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. 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. © 1993 Wiley‐Liss, Inc.