Kelley M R, Jurgens J K, Tentler J, Emanuele N V, Blutt S E, Emanuele M A
Department of Molecular and Cellular Biochemistry, Loyola University of Chicago, Stritch School of Medicine, Maywood.
Alcohol. 1993 May-Jun;10(3):185-9. doi: 10.1016/0741-8329(93)90033-k.
The measurement of alterations in low abundance mRNAs such as the hypothalamic hormones luteinizing hormone-releasing hormone (LHRH) and growth hormone-releasing hormone (GHRH or GRF) from individual hypothalamic tissues in rats has previously been difficult and usually required either isolation of poly(A) mRNA or the pooling of numerous animals to obtain a reasonable signal on Northern blots. Although more sensitive detection methods exist, such as the use of RNA probes or solution hybridization (RNase protection), we have found the most reliable, sensitive, rapid, and accurate method is the reverse transcription-polymerase chain reaction (RT-PCR) using histone H3.3 as an internal control for both steps of this procedure. H3.3 is a cell-cycle independent and constitutively expressed gene in all tissues. We have developed an RT-PCR assay for LHRH and GRF mRNA quantitation and comparative analysis for hypothalamic and extrahypothalamic brain tissues and present the use of RT-PCR for LHRH quantitation in ethanol (EtOH) studies.
以往，测量大鼠个体下丘脑组织中低丰度mRNA的变化，如促黄体激素释放激素（LHRH）和生长激素释放激素（GHRH或GRF）等下丘脑激素，一直很困难，通常需要分离聚腺苷酸（poly(A)）mRNA或汇集大量动物，才能在Northern印迹上获得合理的信号。尽管存在更灵敏的检测方法，如使用RNA探针或溶液杂交（核糖核酸酶保护法），但我们发现，最可靠、灵敏、快速且准确的方法是逆转录聚合酶链反应（RT-PCR），该方法使用组蛋白H3.3作为此过程两个步骤的内参。H3.3是一种不依赖细胞周期且在所有组织中组成性表达的基因。我们开发了一种用于LHRH和GRF mRNA定量以及下丘脑和下丘脑外脑组织比较分析的RT-PCR检测方法，并展示了RT-PCR在乙醇（EtOH）研究中用于LHRH定量的应用。
