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High-resolution quantification of specific mRNA levels in human brain autopsies and biopsies
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Evolutionary Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
2000 (English)In: Genome Research, ISSN 1088-9051, Vol. 10, no 8, 1219-29 p.Article in journal (Refereed) Published
Abstract [en]

Quantification of mRNA levels in human cortical brain biopsies and autopsies was performed using a fluorogenic 5' nuclease assay. The reproducibility of the assay using replica plates was 97%-99%. Relative quantities of mRNA from 16 different genes were evaluated using a statistical approach based on ANCOVA analysis. Comparison of the relative mRNA levels between two groups of samples with different time postmortem revealed unchanged relative expression levels for most genes. Only CYP26A1 mRNA levels showed a significant decrease with prolonged time postmortem (p = 0.00004). Also, there was a general decrease in measured mRNA levels for all genes in autopsies compared to biopsies; however, on comparing mRNA levels after adjusting with reference genes, no significant differences were found between mRNA levels in autopsies and biopsies. This observation indicates that studies of postmortem material can be performed to reveal the relative in vivo mRNA levels of genes. Power calculations were done to determine the number of individuals necessary to detect differences in mRNA levels of 1.5-fold to tenfold using the strategy described here. This analysis showed that samples from at least 50 individuals per group, patients and controls, are required for high-resolution ( approximately twofold changes) differential expression screenings in the human brain. Experiments done on ten individuals per group will result in a resolution of approximately fivefold changes in expression levels. In general, the sensitivity and resolution of any differential expression study will depend on the sample size used and the between-individual variability of the genes analyzed.

Place, publisher, year, edition, pages
2000. Vol. 10, no 8, 1219-29 p.
National Category
Genetics
Identifiers
URN: urn:nbn:se:uu:diva-90900DOI: 10.1101/gr.10.8.1219PubMedID: 10958640OAI: oai:DiVA.org:uu-90900DiVA: diva2:163417
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2010-02-08Bibliographically approved
In thesis
1. Detection of Differentially Expressed Genes in Alzheimer's Disease: Regulator of G-protein Signalling 4: A Novel Mediator of APP Processing
Open this publication in new window or tab >>Detection of Differentially Expressed Genes in Alzheimer's Disease: Regulator of G-protein Signalling 4: A Novel Mediator of APP Processing
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease is a neurodegenerative disease characterised by progressive memory deterioration and cognitive impairment. Pathological hallmarks are extracellular senile plaques, neurofibrillary tangles and neuron loss. Senile plaques are produced through altered processing of the membrane-bound protein APP. Different neurotransmitter signal transduction pathways have been implicated in the formation or development of Alzheimer’s pathologies, but the molecular mechanisms behind these changes are not well known.

The overall aims of this thesis were to identify novel genes with differential expression in Alzheimer’s disease and to investigate mechanisms initiating these changes and their relationship to the disease. A real-time RT-PCR strategy was developed to enable detection of small mRNA changes in human brain autopsy samples. This approach was first used to investigate levels of expression of a candidate gene (MAO), and later employed to verify gene expression differences detected by cDNA microarray analysis. Of several genes verified as differentially expressed in the patients, ITPKB (Inositol 1,4,5-trisphosphate 3-kinase B) and RGS4 (Regulator of G-protein signalling 4) presented the largest expression differences in Alzheimer’s cases compared to control samples. Several splice variants of RGS4 showed similar down-regulation levels and one rare haplotype was associated with decreased RGS4 expression. Functional studies in SH-SY5Y cell cultures overexpressing RGS4 showed that it is likely that RGS4 affects APP processing by regulating PRKC expression levels.

The combined expression of RGS4 and ITPKB is for the first time presented in this thesis as genes with altered mRNA levels in Alzheimer’s disease. These two genes are implicated in the same signalling pathway that modifies calcium levels in the cell. Furthermore, the fact that RGS4 affects APP processing suggests that RGS4 is involved in the development of senile plaques. This motivates further functional studies of this gene and suggests that RGS4 may become a new potential drug target for Alzheimer’s disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 85
Keyword
Alzheimer's disease, RGS4, ITPKB, RAB3A, APP processinig
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-5910 (URN)91-554-6234-X (ISBN)
Public defence
2005-09-29, Ekman-salen, Norby vägen, EBC, 09:15 (English)
Opponent
Supervisors
Available from: 2005-09-08 Created: 2005-09-08 Last updated: 2010-02-08Bibliographically approved
2. High-resolution Studies of mRNA Expression in Brain: A Search for Genes Differently Expressed in Schizophrenia
Open this publication in new window or tab >>High-resolution Studies of mRNA Expression in Brain: A Search for Genes Differently Expressed in Schizophrenia
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene expression differences between patients and controls can be used to find susceptibility genes and drug targets for a disease. High-resolution strategies are required because the differences between the investigated groups may be small and numerous factors may affect the mRNA quantity. This thesis is based on the use of real-time RT-PCR combined with a new statistical approach, developed to detect small differences between patients and controls and differences due to patient subgroups.

Comparisons between human brain biopsy and autopsy samples showed that post-mortem tissue can be used to make conclusions on the relative mRNA levels in the living brain.

Power analysis based on human brain mRNA expression from 14 genes adjusted with two reference genes, revealed that a sample size of 50 patients and 50 controls was required to detect a 2-fold difference with a power and a confidence of 95%. A similar study in rats revealed that approximately the same sample size was required for rat brain mRNA expression studies.

The mRNA levels of several genes were studied in 55 schizophrenia and 55 control prefrontal brain autopsies, using a novel and more powerful statistical analysis. The serotonin receptor 2C gene (HTR2C) showed a significant 1.5-fold decrease in the patients as compared to controls, and the monoamine oxidase B gene (MAOB) a 1.2-fold increase.

The mechanism behind the decrease of HTR2C mRNA levels was investigated by studying the correlation of drug treatment and HTR2C promoter polymorphisms to the HTR2C expression levels. The observed decrease was present in untreated patients, suggesting that the HTR2C mRNA decrease is correlated with the disease and not the treatment. There was no association between promoter polymorphisms and HTR2C expression levels. Thus, the molecular mechanism for the decreased expression remains unclear. Nevertheless, the results support a role for monoaminergic synapses in schizophrenia.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 47 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 894
Keyword
Genetics, mRNA, gene expression, real-time RT-PCR, schizophrenia, 5-HT (serotonin) receptor 2C, brain, psychiatric genetics, Genetik
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-3605 (URN)91-554-5755-X (ISBN)
Public defence
2003-11-07, Lindahlsalen, EBC, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2010-02-08Bibliographically approved

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