通过毛细管电泳结合激光诱导荧光检测和半定量测定小鼠脑中低丰度的胶质纤维酸性蛋白（GFAP）信使核糖核酸（mRNA）

Detection and semi-quantitative determination of low abundance GFAP mRNA in mouse brain by capillary electrophoresis coupled with laser-induced fluorescence.

作者信息

Khan Haseeb Ahmad

机构信息

Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.

出版信息

Brain Res Brain Res Protoc. 2004 Nov;14(1):13-7. doi: 10.1016/j.brainresprot.2004.07.001.

DOI:10.1016/j.brainresprot.2004.07.001

PMID:15519947

Abstract

The sensitivity of capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) was compared with conventional agarose gel electrophoresis-ethidium bromide-UV method (AE-EUV) for detection and semi-quantitative determination of GFAP mRNA in mouse brain. GFAP expression was induced by the neurotoxin MPTP in C57BL mice. Serially diluted RNA samples (0.0003, 0.003, 0.03, 0.3, and 3 microg total RNA) were subjected to RT-PCR and analyzed by both procedures. The integrated pixel density (AE-EUV) and peak area (CE-LIF) were directly proportional to the amount of RNA. However, the observed high sensitivity of CE-LIF suggests its potential application for detection and semi-quantitative determination of low-abundance mRNA transcripts.

摘要

将毛细管电泳与激光诱导荧光检测技术（CE-LIF）的灵敏度，与传统的琼脂糖凝胶电泳-溴化乙锭-紫外光法（AE-EUV）进行了比较，以检测和半定量测定小鼠脑中的胶质纤维酸性蛋白（GFAP）mRNA。在C57BL小鼠中，神经毒素MPTP可诱导GFAP表达。将系列稀释的RNA样品（0.0003、0.003、0.03、0.3和3微克总RNA）进行逆转录聚合酶链反应（RT-PCR），并通过这两种方法进行分析。积分像素密度（AE-EUV）和峰面积（CE-LIF）与RNA量成正比。然而，观察到的CE-LIF的高灵敏度表明其在检测和半定量测定低丰度mRNA转录本方面具有潜在应用价值。