DETECTION OF POLY A+RNA IN SEA-URCHIN EGGS AND EMBRYOS BY QUANTITATIVE INSITU HYBRIDIZATION
DETECTION OF POLY A+RNA IN SEA-URCHIN EGGS AND EMBRYOS BY QUANTITATIVE INSITU HYBRIDIZATION
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DOI：
10.1093/nar/9.12.2819
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发表时间：
1981-01-01
影响因子：
14.9
通讯作者：
ANGERER, RC
中科院分区：
文献类型：
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作者：
ANGERER, LM;ANGERER, RC
我们提出了一种改进的检测~3H聚U杂交法检测聚A区的方法。与Carnoy相比，戊二醛固定法能显著提高RNA的保留率和更好的形态保存性。用蛋白酶K和乙酸酐预杂交处理戊二醛固定的切片，可显著增加信噪比。用溶液滴定法和原位杂交法测量受精后Poly A浓度的增加是非常一致的，表明现场测量产生了对切片中局部RNA浓度的准确的相对估计。对海胆卵和卵裂胚胎切片中颗粒密度分布的研究表明，Poly A+RNA没有主要的细胞质定位，尽管细胞核的标记要少得多，16-细胞胚胎的微粒有少量但显著的PolyA浓度降低。
We present an improved procedure for detecting poly A tractsin situby hybridization of3H poly U. Glutaraldehyde fixation achieves significantly higher retention of RNA and better morphologic preservation than does Carnoy's. A dramatic increase in signal to noise is obtained by prehybridization treatment of glutaraldehyde-fixed sections with proteinase K and acetic anhydride. Measurement of the increase in poly A concentration after fertilization by solution titration and byin situhybridization are in excellent agreement indicating thatin situmeasurements yield accurate relative estimates of local RNA concentrations in sections. Examination of the grain density distribution in sections of sea urchin eggs and cleaving embryos reveals no major cytoplasmic localization of poly A+ RNA, although nuclei show much less labelling and micromeres of 16-cell embryos have a small, but significant, reduction in poly A concentration.