An examination of MS candidate genes identified as differentially regulated in multiple sclerosis plaque tissue, using absolute and comparative real-time Q-PCR analysis

In our laboratory, we have developed methods in real-time detection and quantitative-polymerase chain reaction (Q-PCR) to analyse the relative levels of gene expression in post mortem brain tissues. We have then applied this method to examine differences in gene activity between normal white matter...

Full description

Saved in:

Bibliographic Details
Published in:	Brain research. Brain research protocols Vol. 15; no. 2; pp. 79 - 91
Main Authors:	Tajouri, L., Mellick, A.S., Tourtellotte, A., Nagra, R.M., Griffiths, L.R.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-07-2005
Subjects:	Acute Chronic active Female Gene Dosage Gene Expression Profiling - methods Gene Expression Profiling - standards Gene Expression Regulation Genetic Markers Humans Male Middle Aged Multiple Sclerosis - genetics Multiple Sclerosis - pathology Osteopontin Polymerase Chain Reaction - methods Polymerase Chain Reaction - standards Postmortem Changes Quantitative PCR analysis Reproducibility of Results Osteopontin Quantitative PCR analysis Cellular and molecular biology Chronic active Acute
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	In our laboratory, we have developed methods in real-time detection and quantitative-polymerase chain reaction (Q-PCR) to analyse the relative levels of gene expression in post mortem brain tissues. We have then applied this method to examine differences in gene activity between normal white matter (NWM) and plaque tissue from multiple sclerosis (MS) patients. Genes were selected based on their association with pathology and through identification by previously conducted global gene expression analysis. Plaque tissue was obtained from secondary progressive (SP) patients displaying chronic active, as well as acute pathologies; while NWM from the same location was obtained from age- and sex-matched controls (normal patients). In this study, we used both SYBR Green I supplementation and commercially available mixes to assess both comparative and absolute levels of gene activity. The results of both methods compared favourably for four of the five genes examined ( P < 0.05, Pearsons), while differences in gene expression between chronic active and acute pathologies were also identified. For example, a >50-fold increase in osteopontin ( Spp1) and inositol 1-4-5 phosphate 3 kinase B ( Itpκb) levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques ( P < 0.05, unpaired t test). By contrast, there was no significant difference in the levels of the MS marker and calcium-dependent protease (Calpain, Capns1) in MS plaque tissue. In summary, Q-PCR analysis using SYBR Green I has allowed us to economically obtain what may be clinically significant information from small amounts of the CNS, providing an opportunity for further clinical investigations.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1385-299X
DOI:	10.1016/j.brainresprot.2005.04.003