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Dahlgren, Andreas
Publications (2 of 2) Show all publications
Liljedahl, U., Fredriksson, M., Dahlgren, A. & Syvänen, A.-C. (2004). Detecting imbalanced expression of SNP alleles by minisequencing on microarrays. BMC Biotechnology, 4(24), 1-10
Open this publication in new window or tab >>Detecting imbalanced expression of SNP alleles by minisequencing on microarrays
2004 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 4, no 24, p. 1-10Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs) may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system.

RESULTS:

The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1-9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and minisequencing in a microtiter plate format.

CONCLUSIONS:

We conclude that microarray based minisequencing is an accurate and accessible tool for multiplexed screening for imbalanced allelic expression in multiple samples and tissues in parallel.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-92628 (URN)10.1186/1472-6750-4-24 (DOI)000225233200001 ()15500681 (PubMedID)
Available from: 2005-02-18 Created: 2005-02-18 Last updated: 2017-12-14Bibliographically approved
Silander, K., Axelsson, T., Widén, E., Dahlgren, A., Palotie, A. & Syvänen, A.-C. (2003). Analysis of genetic variation in the GenomEUtwin project.. In: .
Open this publication in new window or tab >>Analysis of genetic variation in the GenomEUtwin project.
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2003 (English)Chapter in book (Other scientific)
Keywords
Databases; Genetic, European Union, Genotype, Humans, International Cooperation, Microsatellite Repeats, Polymorphism; Single Nucleotide, Twin Studies, Variation (Genetics)/*genetics
Identifiers
urn:nbn:se:uu:diva-76884 (URN)14624723 (PubMedID)
Available from: 2006-10-30 Created: 2006-10-30
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