This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated by denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 sec, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the reproducible, statistically significant reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of the poly(A) binding protein, PABPC1, with polyadenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, and cost-effective method to enable researchers to analyze expression, processing, binding, and decay of RNAs.

中文翻译：

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Chemi-Northern：一种用于 RNA 分子分析和定量的多功能化学发光 Northern 印迹方法

本报告描述了一种基于化学发光的 RNA 印迹检测方法，称为 Chemi-Northern。这种方法建立在 Northern 印迹的简单性和多功能性的基础上，同时无需昂贵且繁琐的放射性。首先通过变性凝胶电泳分离 RNA，转移到尼龙膜上，然后与生物素化的 RNA 或 DNA 反义探针杂交。然后使用与辣根过氧化物酶缀合的链霉亲和素和增强化学发光底物来检测与靶RNA结合的探针。我们的结果证明了该方法在检测细胞中表达的天然和工程 RNA（包括信使 RNA 和非编码 RNA）方面的多功能性。我们证明 Chemi-Northern 检测灵敏且快速，可在短短 1 秒内检测出阿托摩尔量的 RNA，且信号强度高、背景低。动态响应表现出出色的线性度。使用 Chemi-Northern，我们测量了人类序列特异性 RNA 结合蛋白 PUM1 和 PUM2 对 mRNA 水平的可重复的、统计上显着的降低。此外，我们还测量了聚腺苷酸结合蛋白 PABPC1 与聚腺苷酸化 mRNA 的相互作用。因此，Chemi-Northern 方法提供了一种通用、简单且经济高效的方法，使研究人员能够分析 RNA 的表达、加工、结合和衰变。