Gainer Harold, Ponzio Todd A, Yue Chunmei, Kawasaki Makoto
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2011;789:89-110. doi: 10.1007/978-1-61779-310-3_5.
Measurements of changes in pre-mRNA levels by intron-specific probes are generally accepted as more closely reflecting changes in gene transcription rates than are measurements of mRNA levels by exonic probes. This is, in part, because the pre-mRNAs, which include the primary transcript and various splicing intermediates located in the nucleus (also referred to as heteronuclear RNAs, or hnRNAs), are processed rapidly (with half-lives <60 min) as compared to neuropeptide mRNAs, which are then transferred to the cytoplasm and which have much longer half-lives (often over days). In this chapter, we describe the use of exon-and intron-specific probes to evaluate oxytocin (OT) and vasopressin (VP) neuropeptide gene expression by analyses of their mRNAs and hnRNAs by quantitative in situ hybridization (qISH) and also by using specific PCR primers in quantitative, real-time PCR (qPCR) procedures.
与外显子探针测量mRNA水平相比，用内含子特异性探针测量前体mRNA水平的变化通常被认为更能准确反映基因转录速率的变化。部分原因在于，前体mRNA（包括位于细胞核中的初级转录本和各种剪接中间体，也称为异核RNA或hnRNA）与神经肽mRNA相比，加工速度很快（半衰期<60分钟），神经肽mRNA随后转移到细胞质中，半衰期长得多（通常超过数天）。在本章中，我们描述了通过定量原位杂交（qISH）分析其mRNA和hnRNA，以及在定量实时PCR（qPCR）程序中使用特异性PCR引物，利用外显子和内含子特异性探针来评估催产素（OT）和加压素（VP）神经肽基因的表达。
