BACKGROUND: A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules. RESULTS: The sensitivity of the assay was approximately 1.2 x 109 (2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities. CONCLUSIONS: The here presented sandwich hybridisation method was succefully applied for monitoring the amounts of ribosomal RNA and mRNA with high expression level in shake flask cultivation conditions. Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions.

中文翻译：

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三明治杂交测定法定量检测粗细胞裂解物中的酵母RNA。

背景：快速的基于微量滴定板的夹心杂交测定法被开发用于使用磁珠检测和定量单个RNA种类。溶液杂交后，通过生物素-链霉亲和素亲和力结合收集靶RNA分子，并通过碱性磷酸酶产生的荧光信号进行检测。酿酒酵母的18S rRNA和SUC2 mRNA被用作模型RNA靶分子。结果：该测定的灵敏度约为目标RNA的1.2 x 109（2 fmol）分子。所开发的方法对于啤酒酵母的粗制细胞裂解物是可行的，并且通过在抑制和诱导条件下测量细胞生长过程中18S rRNA的水平以及SUC2 mRNA进行评估。18S rRNA表达水平遵循特定增长率的变化。SUC2 mRNA水平与测得的转化酶活性高度相关。结论：本文提出的三明治杂交方法成功地用于监测摇瓶培养条件下高表达水平的核糖体RNA和mRNA的量。夹心杂交方法为在生产条件下追踪感兴趣的单个关键RNA物种提供了快速便捷的工具。