DETECTION OF MESSENGER-RNAS IN SEA-URCHIN EMBRYOS BY INSITU HYBRIDIZATION USING ASYMMETRIC RNA PROBES
DETECTION OF MESSENGER-RNAS IN SEA-URCHIN EMBRYOS BY INSITU HYBRIDIZATION USING ASYMMETRIC RNA PROBES
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DOI：
10.1016/0012-1606(84)90162-3
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发表时间：
1984-01-01
影响因子：
2.7
通讯作者：
ANGERER, RC
中科院分区：
文献类型：
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作者：
COX, KH;DELEON, DV;ANGERER, RC
不对称RNA探针只含有编码mRNA的链，与使用对称(双链都有)的RNA或DNA探针相比，原位杂交效率有很大提高。不对称RNA探针是通过从编码海胆mRNA的序列和转录载体R7β7之间形成的重组体转录而在体外合成的。用代表早期变异组蛋白mRNA序列的探针与海胆胚胎切片杂交，研究了杂交产物的热稳定性、最适温度、序列差异对杂交热稳定性的影响以及杂交信号对探针浓度和杂交时间的依赖关系。从观察到的信号估计表明，很大一部分目标RNA被保留在片段中，并在饱和时与探针杂交。再加上异源探针非特异性背景结合的测量，这些数据表明该方法具有足够的灵敏度来检测许多中等丰度的mRNA(在1500个细胞的Pluteus中每个细胞20-75个分子)。对不同发育阶段的胚胎进行原位杂交，结果表明，组蛋白mRNA在卵裂胚胎中的分布较为均匀，而不同细胞系的老年胚胎在组蛋白mRNA的积累上存在较大差异。
Asymmetric RNA probes, which contain only the mRNA coding strand, provide a large increase in hybridization efficiency in situ over that observed with either symmetric (both strands represented) RNA or DNA probes. Asymmetric RNA probes are synthesized in vitro by transcription from recombinants formed between sequences encoding sea urchin mRNA and the transcription vector R7.DELTA.7. Using a probe representing early variant histone mRNA sequences, hybridization to sections of sea urchin embryos was characterized with respect to thermal stability of the hybrids formed, optimum temperature, effect of sequence divergence in hybrid thermal stability, and dependence of the hybridization signals on probe concentration and hybridization time. Estimates from the observed signals indicate that a large fraction of target RNA is retained in sections and hybridized with probe at saturation. Coupled with measurements of nonspecific background binding of heterologous probes, these data indicate that the method has sufficient sensitivity to detect many moderately abundant mRNA (20-75 molecules per cell in the 1500-cell pluteus). In situ hybridization to embryos at different developmental stages shows that while histone mRNA are uniformly distributed to cleaving embryos, different cell lineages of older embryos show large differences in accumulation of these mRNA.