Project description:Genotyping was performed using a custom-designed Illumina 384-SNP VeraCode microarray (Illumina) to determine possible associations of genes encoding PADI1, PADI2, PADI3, PADI4, PADI6, PRKD3, Gc, GLRX,CDSN, PSORS1C1, TXNDC5, CA1, bub3, SORBS1, VDR,SERPNA1, PCSK6, DNAH9 to rheumatoid arthrities (RA) and ankylosing spondylitis (AS). We previously found that encoding genes of PADI1, PADI2, PADI3, PADI4, PADI6, PRKD3, Gc, GLRX,CDSN, PSORS1C1, TXNDC5, CA1, bub3, SORBS1, VDR, SERPNA1, PCSK6 and DNAH9 could be associated with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). We used illumina customer designed microarray to verify the above finding with an increased numbers of samples. Genotyping was performed using a custom-designed Illumina 384-SNP VeraCode microarray (Illumina) to determine possible associations of genes encoding PADI1, PADI2, PADI3, PADI4, PADI6, PRKD3, Gc, GLRX,CDSN, PSORS1C1, TXNDC5, CA1, bub3, SORBS1, VDR, SERPNA1, PCSK6, DNAH9 to RA and AS. A case-control study was performed with 51 AS patients, 266 rheumatoid arthritis (RA) and 163 health controls. Tag single nucleotide polymorphisms (tag SNPs) across the above genes were determined by searching the HapMap database. The tag SNPs were selected on the basis of linkage disequilibrium patterns observed in the Han Chinese in Beijing (CHB) samples that were genotyped as a part of the International HapMap Project. Only SNPs with a minor allele frequency of greater than 5% with a pair-wise r2 ≥ 0.8 in the HapMap were considered. SNPs that were in exons and untranslated regions (UTR), as well as promoters and introns within 500 bp of exons were also selected. Candidate SNPs were submitted to Illumina for a design score. The Illumina Assay Design Tool filtered out SNPs that were not suitable for the Illumina platform. Finally, 382 SNPs with a design score of “1” were selected. 382 SNPs within PADI1, PADI2, PADI3, PADI4, PADI6, PRKD3, Gc, GLRX,CDSN, PSORS1C1, TXNDC5, CA1, bub3, SORBS1, VDR,SERPNA1, PCSK6, DNAH9 encoding genes were selected. Genotyping was performed using Illumina VeraCode microarray in a case-control study including 51 AS patients, 266 rheumatoid arthritis (RA) and 163 health controls We performed genotyping using a custom-designed Illumina 384-SNP VeraCode microarray (Illumina). Peripheral blood samples were collected from patients with RA (n=266, 183 female) and AS (n=51, 10 female). RA patients had a mean age of 51.7 years, while AS patients had a mean age of 35.9 years. A total of 163 (60 female) healthy individuals with a mean age of 48.0 years were blood donors. Blood samples were put into Monovette tubes containing 3.8% sodium citrate. The genotyping was conducted with the BeadXpress Reader using the Illumina VeraCode GoldenGate Assay kit. 500 ng of sample DNA were used per assay. Genotype clustering and calling were performed using BeadStudio software (Genomestudio V2010.2 Genotyping module V1.6) (Illumina). This work was completed at the Beijing Institute of Genomics.

Project description:The Rosa-rtTAnls/tetO-HoxA10 mouse model is a Tetracycline-On (tetO) system in which the expression level of HoxA10 can be tightly regulated and overexpressed under administration of the Doxycycline (DOXY) in the drinking bottle of mice or fed with Ciproxine (CIPRO) as a negative control of HoxA10 expression. In this experiment, we analyzed expressed genes derived from microarray analysis of HoxA10 Acute Myeloid Leukemia (AML) cells induced in primary recipient mice (1ary) and in secondary recipient mice, with HoxA10 overexpression (DOXY) or after turning off the HoxA10 overexpression (CIPRO). Concerning the biological design, we compared leukemia induced in primary recipient mice (1ary) to normal bone marrow (normal BM). We compared also primary leukemia (1ary) to leukemia induced in secondary recipient mice (2ary) induce by continuous expression of HoxA10 (DOXY) to leukemia induced in secondary recipient mice after turning off expression of HoxA10 (CIPRO) and analyse relapse growth of leukemic cells after turning off HoxA10. Concerning the protocol, the leukemic cells originated from two independent primary AML donor mice (cl1, cl2, cl3, cl4) are injected in secondary recipient mice, which are monitored for reoccurrence of leukemia. AML are induced by Doxycycline (Doxy) or Ciproxine as negative control (Cipro), by administration of 0.2g/mL continuously in the drinking bottle until development of AML. In sick mice, femurs and tibias were removed and cleaned of soft tissue, bones were crushed, and the cell suspension was filtered over a 100-m pore size cell strainer filter (Falcon; Becton Dickinson). Total RNA from BM cells of ciproxine and doxycycline receiving recipient mice suffering from leukemia was extracted from bone marrow cells (Qiagen). RNA concentration and integrity were respectively determined by NanoDrop spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology). Reverse transcription using Illumina TotalPrep RNA Amplification Kit (Ambion) and hybridization to Mouse WG-6 Expression v1.1 BeadChips (Illumina) interrogating more than 45,000 transcripts were performed by the Swegene Center for Integrative Biology at Lund University (SCIBLU).

Project description:Illumina Infinium whole genome genotyping (WGG) arrays are increasingly being applied in cancer genomics to study gene copy number alterations and allele-specific aberrations such as loss-of-heterozygosity (LOH). Methods developed for normalization of WGG arrays have mostly focused on diploid, normal samples. However, for cancer samples genomic aberrations may confound normalization and data interpretation. Therefore, we examined the effects of the conventionally used normalization method for Illumina Infinium arrays when applied to cancer samples. We demonstrate an asymmetry in the detection of the two alleles for each SNP, which deleteriously influences both allelic proportions and copy number estimates. The asymmetry is caused by a remaining bias between the two dyes used in the Infinium II assay after using the normalization method in Illumina’s proprietary software (BeadStudio). We propose a quantile normalization strategy for correction of this dye bias. We tested the normalization strategy using 535 individual hybridizations from 10 data sets from the analysis of cancer genomes and normal blood samples generated on Illumina Infinium II 300k version 1 and 2, 370k and 550k BeadChips. We show that the proposed normalization strategy successfully removes asymmetry in estimates of both allelic proportions and copy numbers. Additionally, the normalization strategy reduces the technical variation for copy number estimates while retaining the response to copy number alterations. The proposed normalization strategy represents a valuable low-level analysis tool that improves the quality of data obtained from Illumina Infinium arrays, in particular when used for LOH and copy number variation studies. To investigate the effects of a quantile normalization of Illumina Infinium data, compared to conventional normalization using BeadStudio (www.illumina.com), we renormalized 535 individual hybridizations conducted on Illumina 300K, 370K and 550K BeadChips. Sample types included breast cancer, colon cancer, urothelial carcinoma, leukemia as well as normal blood and HapMap samples. This series includes the 6 breast cancers hybridized on Illumina HumanHap 550K BeadChips.

Project description:The Rosa-rtTAnls/tetO-HoxA10 mouse model is a Tetracycline-On (tetO) system in which the expression level of HoxA10 can be tightly regulated and overexpressed under administration of the Doxycycline (Doxy) in the drinking bottle of mice or fed with Ciproxine (Cipro) as a negative control of HoxA10 expression. In this experiment, we analyzed expressed transcripts derived from microarray analysis of HoxA10 Acute Myeloid Leukemia (AML) cells induced in secondary recipient mice, with HoxA10 overexpression (Doxy) or after turning off the HoxA10 overexpression (Cipro). Concerning the biological design, we compared leukemia in secondary recipient mice induced by continuous expression of HoxA10 (Doxy) to leukemia induced after turning off expression of HoxA10 (Cipro) and analyse relapse growth of leukemic cells after turning off HoxA10. Concerning the protocol, the leukemic cells originated from two independent primary AML donor mice (cl3 and cl4) are injected in secondary recipient mice, which are monitored for reoccurrence of leukemia. AML are induced by Doxycycline (Doxy) or Ciproxine as negative control (Cipro), by administration of 0.2g/mL continuously in the drinking bottle until development of AML. In sick mice, femurs and tibias were removed and cleaned of soft tissue, bones were crushed, and the cell suspension was filtered over a 100-m pore size cell strainer filter (Falcon; Becton Dickinson). Total DNA from BM cells of Cipro and Doxy receiving recipient mice suffering from leukemia was extracted from bone marrow cells (Qiagen). DNA concentration and integrity were respectively determined by NanoDrop spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology). Hybridization to Mouse CGH microarray 244A (Agilent) were performed by the Atlas Biolabs (Germany). Reverse transcription using Illumina TotalPrep RNA Amplification Kit (Ambion) and hybridization to Mouse WG-6 Expression v2 BeadChips (Illumina) were performed by the Swegene Center for Integrative Biology at Lund University (SCIBLU).

Project description:The Rosa-rtTAnls/tetO-HoxA10 mouse model is a Tetracycline-On (tetO) system in which the expression level of HoxA10 can be tightly regulated and overexpressed under administration of the Doxycycline (Doxy) in the drinking bottle of mice or fed with Ciproxine (Cipro) as a negative control of HoxA10 expression. In this experiment, we analyzed expressed transcripts derived from microarray analysis of HoxA10 Acute Myeloid Leukemia (AML) cells induced in secondary recipient mice, with HoxA10 overexpression (Doxy) or after turning off the HoxA10 overexpression (Cipro). Concerning the biological design, we compared leukemia in secondary recipient mice induced by continuous expression of HoxA10 (Doxy) to leukemia induced after turning off expression of HoxA10 (Cipro) and analyse relapse growth of leukemic cells after turning off HoxA10. Concerning the protocol, the leukemic cells originated from two independent primary AML donor mice (cl1 and cl2) are injected in secondary recipient mice, which are monitored for reoccurrence of leukemia. AML are induced by Doxycycline (Doxy) or Ciproxine as negative control (Cipro), by administration of 0.2g/mL continuously in the drinking bottle until development of AML. In sick mice, femurs and tibias were removed and cleaned of soft tissue, bones were crushed, and the cell suspension was filtered over a 100-m pore size cell strainer filter (Falcon; Becton Dickinson). Total DNA from BM cells of Cipro and Doxy receiving recipient mice suffering from leukemia was extracted from bone marrow cells (Qiagen). DNA concentration and integrity were respectively determined by NanoDrop spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology). Hybridization to Mouse CGH microarray 244A (Agilent) were performed by the Atlas Biolabs (Germany). Reverse transcription using Illumina TotalPrep RNA Amplification Kit (Ambion) and hybridization to Mouse WG-6 Expression v1.1 BeadChips (Illumina) were performed by the Swegene Center for Integrative Biology at Lund University (SCIBLU).

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Expression profile and whole genome SNP data from acute monocytic leukemia patients

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