Abstract
Evaluation of messenger RNA (mRNA) expression has long been used as evidence demonstrating alterations in gene expression at the transcriptional level following global ischemia. Given the complexity of focal cerebral ischemic injury, in situ analysis of the localization and coexpression of specific RNAs is necessary to tease apart differentially susceptible populations of neurons to ischemic injury. Historically, RNA has been long characterized by its role as the “messenger in the middle” between DNA and proteins, where it was seen as primarily a stopping ground and potential added layer of control of protein expression. However, in recent years, RNA has begun to be understood as much more than simply a messenger. The discovery of microRNA (miRNA) and other small noncoding RNA species has led to a reemergence of RNA-based applications, particularly in the investigation of pathological conditions, such as focal cerebral ischemia. One of the strongest of these techniques lies in the modification of polymerase chain reaction (PCR). In this chapter, we present the basic techniques of reverse transcription-PCR for mRNA and miRNA detection and DIG-labeled oligonucleotide in situ hybridization in the context of focal cerebral injury.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Akins PT, Liu PK, Hsu CY (1996) Immediate early gene expression in response to cerebral ischemia. Friend or foe? Stroke 27:1682–1687
Sharp FR, Lu A, Tang Y, Millhorn DE (2000) Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab 19:1247–1255
Shin YJ, Choi JS, Choi JY, Cha JH, Chun MH, Lee MY (2010) Enhanced expression of vascular endothelial growth factor receptor-3 in the subventricular zone of stroke-lesioned rats. Neurosci Lett 469:194–198
Ding Y, Xu L, Jovanovic BD, Helenowski IB, Kelly DL, Catalona WJ, Yang XJ, Pins M, Bergan RC (2007) The methodology use to measure differential gene expression affects the outcome. J Biomol Tech 18:321–330
Murphy J, Bustin SA (2009) Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard? Expert Rev Mol Diagn 9:187–197
Meister G (2007) miRNAs get an early start on translational silencing. Cell 131:25–28
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20
Jeyaseelan K, Lim KY, Armugam A (2008) MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke 39:959–966
Dharap A, Bowen K, Place R, Li LC, Vemuganti R (2009) Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome. J Cereb Blood Flow Metab 29:675–687
Yin KJ, Deng Z, Huang H, Hamblin M, Xie C, Zhang J, Chen YE (2010) miR-497 regulates neuronal death in mouse brain after transient focal cerebral ischemia. Neurobiol Dis 38:17–26
Kiyama H, Emson PC, Tohyama M (1990) Recent progress in the use of the technique of non-radioactive in situ hybridization histochemistry: new tools for molecular neurobiology. Neurosci Res 9:1–21
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gan, Y., Stetler, R.A. (2012). Biochemical and Molecular Biological Assessments of Focal Cerebral Ischemia: mRNA and MicroRNA. In: Chen, J., Xu, XM., Xu, Z., Zhang, J. (eds) Animal Models of Acute Neurological Injuries II. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1007/978-1-61779-782-8_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-782-8_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-781-1
Online ISBN: 978-1-61779-782-8
eBook Packages: Springer Protocols