Quantitative analysis of in situ hybridization methods for the detection of actin gene expression.
Quantitative analysis of in situ hybridization methods for the detection of actin gene expression.
复制标题
用于检测肌动蛋白基因表达的原位杂交方法的定量分析。
DOI：
10.1093/nar/13.5.1777
复制
发表时间：
1985
影响因子：
14.9
通讯作者：
Singer,RH
中科院分区：
文献类型：
--
作者：
Lawrence,JB;Singer,RH
我们已经实现了一个有效的，定量的方法优化ofin situhybridization使用双链重组DNA探针。本研究以鸡胚肌肉组织中肌动蛋白mRNA的表达为模型。肌动蛋白和对照（pBR 322）探针与P32标记的核苷酸切口平移，与盖玻片上生长的细胞杂交，并在闪烁计数器中定量。通过在细胞固定之前将H3-尿苷掺入RNA中来监测细胞RNA保留。一千多个样品进行了分析，并检查的技术变量中的固定协议，蛋白水解细胞的预处理，杂交的时间过程中，饱和动力学，杂交效率，和探针大小对杂交和网络形成的影响。结果使我们能够开发一种可重复的原位杂交方法，该方法比其他方法更简单，对细胞RNA和形态的破坏性更小。此外，该技术在检测细胞RNA方面是高度灵敏和有效的。最后，用于该分析的快速定量方法本身作为过滤或溶液杂交的潜在替代方案是有价值的。
We have implemented an efficient, quantitative approach for the optimization ofin situhybridization using double-stranded recombinant DNA probes. The model system studied was actin mRNA expression in chicken embryonic muscle cultures. Actin and control (pBR322) probes were nick-translated with P32labeled nucleotides, hybridized to cells grown on coverslips, and quantitated in a scintillation counter. Cellular RNA retention was monitored via the incorporation of H3-Uridine into RNA prior to cell fixation. Over a thousand samples were analyzed, and among the technical variables examined were the fixation protocol, proteolytic cell pretreatment, the time course of hybridization, saturation kinetics, hybridization efficiency, and effect of probe size on hybridization and network formation. Results have allowed us to develop a reproduciblein situhybridization methodology which is simpler and less destructive to cellular RNA and morphology than other protocols. Moreover, this technique is highly sensitive and efficient in detection of cellular RNAs. Lastly, the rapid quantitative approach used for this analysis is valuable in itself as a potential alternative to filter or solution hybridizations.