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首页> 外文期刊>Brain research. Brain research protocols >Simultaneous determination of multiple transcripts and splice variants of a primary transcript using ribonuclease protection assays.
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Simultaneous determination of multiple transcripts and splice variants of a primary transcript using ribonuclease protection assays.

机译：使用核糖核酸酶保护试验同时测定初级转录本的多个转录本和剪接变体。

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The ribonuclease (RNase) protection assay (RPA) is an extremely sensitive technique used to determine specific mRNAs from cell and tissue extracts. The present protocol presents detailed procedures for a conventional RPA using antisense RNA probes purified with a Fullengther apparatus. The Fullengther has the advantage of being a relatively quick and safe procedure compared to more conventional methods for purification of full-length RNA probes. Using this protocol, we sought to simultaneously determine multiple mRNA species, including splice variants of the type I receptor (PAC(1)) of pituitary adenylate cyclase-activating polypeptide (PACAP), an important mediator in the regulation of luteinizing hormone-releasing hormone (LHRH) synthesis by ovarian steroids such as progesterone [7]. PAC(1) has more than eight splice variants. We have been able to discriminate the hop1 variant from other splice variants. To improve our understanding of the regulation mechanism of genes that are related to each other, such as LHRH and PACAP, it is most important to simultaneously determine genes that are involved in the same physiological areas of regulation. Using only 5 microg of total RNA sample from a single rat preoptic area, we simultaneously determined five different transcripts, including four rare mRNA species such as LHRH, PACAP, and hop1 variant and other splice variants of PAC(1), as well as the internal control of cyclophilin mRNA. This protocol provides a method for the simultaneous determination of multiple transcripts using the RPA.
机译：核糖核酸酶（RNase）保护测定（RPA）是一种非常灵敏的技术，用于从细胞和组织提取物中确定特定的mRNA。本协议提供了常规的RPA的详细程序，该常规RPA使用的是用Fullengther仪器纯化的反义RNA探针。与更常规的纯化全长RNA探针的方法相比，Fullengther具有相对快速和安全的操作优势。使用此协议，我们试图同时确定多个mRNA物种，包括垂体腺苷酸环化酶激活多肽（PACAP）的I型受体（PAC（1））的剪接变体，它是调节促黄体激素释放激素的重要介体（LHRH）由卵巢类固醇如孕酮合成[7]。 PAC（1）具有八个以上的剪接变体。我们已经能够将Hop1变体与其他剪接变体区分开。为了增进我们对彼此相关的基因（例如LHRH和PACAP）的调控机制的了解，最重要的是同时确定涉及同一生理调控区域的基因。仅使用来自单个大鼠视前区的5微克总RNA样品，我们同时确定了五种不同的转录本，包括四种罕见的mRNA种类，如LHRH，PACAP和Hop1变体以及PAC（1）的其他剪接变体，以及亲环蛋白mRNA的内部控制。该协议提供了一种使用RPA同时确定多个转录本的方法。

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