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Detecting imbalanced expression of SNP alleles by minisequencing on microarrays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
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.

Place, publisher, year, edition, pages
2004. Vol. 4, no 24, p. 1-10
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-92628DOI: 10.1186/1472-6750-4-24ISI: 000225233200001PubMedID: 15500681OAI: oai:DiVA.org:uu-92628DiVA, id: diva2:165777
Available from: 2005-02-18 Created: 2005-02-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Using Minisequencing Technology for Analysing Genetic Variation in DNA and RNA
Open this publication in new window or tab >>Using Minisequencing Technology for Analysing Genetic Variation in DNA and RNA
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.

The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. p. 55
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 10
Keywords
Molecular medicine, microarray, minisequencing, molecular medicine, single nucleotide polymorphism, stem cell transplantation, whole genome amplification, allelic imbalance, alternative splicing, Molekylärmedicin
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-4789 (URN)91-554-6152-2 (ISBN)
Public defence
2005-03-15, Rudbecksalen, Rudbecklaboratoriet, Uppsala, 09:15
Opponent
Supervisors
Available from: 2005-02-18 Created: 2005-02-18 Last updated: 2018-01-13Bibliographically approved
2. Microarray Technology for Genotyping in Pharmacogenetics
Open this publication in new window or tab >>Microarray Technology for Genotyping in Pharmacogenetics
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The studies in this thesis describe the development of a microarray based minisequencing system and its application to highly parallel genotyping of single nucleotide polymorphisms. The technical developments included identification of a three-dimensional microarray surface coating with high binding capacity for oligonucleotides modified with amino groups as the most optimal one for the system. The system was also established for multiplexed, reproducible quantitative analysis of SNP alleles both on the level of DNA and RNA. The sensitivity of the system to distinguish SNP alleles present as a minority in a mixed sample was found to be 1-6%.

The microarray based minisequencing system was applied in a pharmacogenetic study on antihypertensive drug response. A panel of 74 SNPs located in candidate genes related to blood pressure regulation were genotyped in DNA samples from hypertensive patients that had been treated with the antihypertensive drugs irbesartan or atenolol. Multiple regression analysis of the genotype data against the reduction in blood pressure identified genotype combinations of four to five SNPs that explain 44-56% of the reduction in blood pressure in the two treatment groups. The genotypes of two individual SNPs in the angiotensinogen (AGT) gene and a SNP in the low density lipoprotein receptor (LDLR) gene appeared to be associated to reduced blood pressure after treatment with atenolol, while a SNP in the apolipoprotein B (APOB) gene was associated to blood pressure reduction after irbesartan treatment. The genotype of one SNP in the adrenergic alpha-2A-receptor gene (ADRA2A) was related to the reduction in left ventricular mass following atenolol treatment while the genotypes of two SNPs, one in the APOB gene and one in the AGT gene were related to the reduction in left ventricular mass in the patients treated with irbesartan.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. p. 69
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1342
Keywords
Molecular medicine, microarray, genotyping, pharmacogenetics, molecular medicine, single nucleotide polymorphism, hypertension, Molekylärmedicin
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-4222 (URN)91-554-5937-4 (ISBN)
Public defence
2004-05-13, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15
Opponent
Supervisors
Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2018-01-13Bibliographically approved

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Liljedahl, UlrikaDahlgren, AndreasSyvänen, Ann-Christine

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