Analysis of multiple exon-skipping mRNA splice variants using SYBR Green real-time RT-PCR

Walton, Heather S., Gebhardt, Florian M., Innes, David J. and Dodd, Peter R. (2007) Analysis of multiple exon-skipping mRNA splice variants using SYBR Green real-time RT-PCR. Journal of Neuroscience Methods, 160 2: 294-301.


Author Walton, Heather S.
Gebhardt, Florian M.
Innes, David J.
Dodd, Peter R.
Title Analysis of multiple exon-skipping mRNA splice variants using SYBR Green real-time RT-PCR
Journal name Journal of Neuroscience Methods   Check publisher's open access policy
ISSN 0165-0270
Publication date 2007
Year available 2006
Sub-type Article (original research)
DOI 10.1016/j.jneumeth.2006.09.022
Volume 160
Issue 2
Start page 294
End page 301
Total pages 8
Place of publication Amsterdam
Publisher Elsevier Science Bv
Collection year 2008
Language eng
Subject 270201 Gene Expression
320799 Neurosciences not elsewhere classified
C1
730203 Health related to ageing
Abstract Fluorescence-based PCR techniques are becoming an increasingly popular method for measuring low-abundance alternatively spliced mRNA transcripts. The dynamic range of real-time RT-PCR affords high sensitivity for the measurement of gene expression, but this mandates the need for strict controls to ensure assay validity. Primer design, reverse transcription, and cycling conditions need to be optimized to ensure an accurate and reproducible assay. Here, we describe a procedure for creating a cost effective and reliable method for the absolute quantification of several exon-skipping variants of human excitatory amino acid transporter-2 (EAAT2). We show that the cycling conditions can be adjusted to increase the specificity of primers that span exon-exon junctions, and that differences in the reverse transcription reaction can be minimized. Standard curves are stable and produce accurate absolute copy number data. We report that exon-skipping transcripts, EAAT2 Delta 7 and EAAT2 Delta 9, account for 5.8% of EAAT2 mRNA in autopsy human neocortex. (c) 2006 Elsevier B.V. All rights reserved.
Keyword Biochemical Research Methods
Neurosciences
glutamate transporter
absolute quantification
EAAT2
human brain
alternative splicing
Polymerase-chain-reaction
Metabolically Active Synaptosomes
Amyotrophic-lateral-sclerosis
Glutamate Transporter Eaat2
Human-brain
Targeted Amplification
Frozen Rat
Quantification
Genes
Assay
Q-Index Code C1
Q-Index Status Confirmed Code

 
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Created: Mon, 18 Feb 2008, 16:36:49 EST