Institutionen för medicinska vetenskaper

Array och analysfaciliteten

Välkommen till Array och analysfaciliteten!

Plattformen fungerar som en serviceenhet som tillhandahåller mikroarrayteknologi till forskare främst inom akademin, men även till externa intressenter. Här finner du information om vilka applikationer från Affymetrix och vilken bioinformatisk support vi på Array och analysfaciliteten erbjuder.

Kontakta oss gärna via arrayplatform[at]medsci.uu.se eller 018-4714484 för ett möte om Ni planerar ett projekt, så hjälper vi gärna till för att se till att upplägget blir det bästa möjliga! Se nedan för information om priser för våra analyser och de krav som ställs på prover gällande kvalitet och kvantitet.

Läs mer om olika Applikationer, vår Bioinformatiksupport, Protokoll och provberedning och den Forskning och utveckling vi bedriver.

Array och analysfaciliteten är ett samarbete mellan Uppsala universitet och Akademiska laboratoriet.

Applikationer

The Affymetrix technology

GeneChip probe arrays are manufactured using technology that combines photolithography and combinatorial chemistry.

Expression analysis

- Whole-Transcript Gene Expression & Alternative Splicing

Affymetrix whole-transcript gene expression and alternative splicing solutions allow you to measure gene expression at the transcript and exon level.

- 3' Gene Expression

The GeneChip® Human Genome U133 Plus 2.0 Array offers comprehensive analysis of genome-wide expression on a single array. Eleven pairs of oligonucleotide probes are used to measure the level of transcription of each sequence represented on the array.

 - miRNA

 - Formalin fixed paraffin embedded samples

This method provides the latest and most robust RNA amplification and labeling solution for whole-genome expression profiling from FFPE samples.

Expression assays can be run on cartridge arrays or on our new system:

GeneTitan® Multi-Channel (MC) Instrument

The GeneTitan Instrument automates array processing from target hybridization to data generation by combining a hybridization oven, fluidics processing, and state-of-the art imaging device into a single bench-top instrument.

Genotyping

- Axiom Genotyping

Axiom Genotyping Solution includes pre-designed and customized array plates with validated genomic content from the Axiom Genomic Database as well as complete reagent kits, data analysis tools, and a fully automated workflow utilizing GeneTitan® Multi-Channel (MC) Instrument.

Copy number analysis

- Fresh or fresh frozen samples

The GeneChip SNP arrays are dual-purpose arrays, simultaneously providing both copy number predictions and genotypes for loss of heterozygosity (LOH) determination.

The high density of markers on these arrays enables identification of gains and losses with increased confidence because of the many data points predicting even the small copy number aberrations. The availability of genotyping data makes the detection of LOH possible.

 - Formalin fixed paraffin embedded samples

A new exciting possibility is to study allele-specific genomic copy numbers using Formalin Fixed Paraffin Embedded (FFPE) samples. Until recently fresh or fresh frozen samples have been required for genomic copy number analysis using SNP arrays. With the introduction of OncoScan™ technology based on molecular inversion probes also the many FFPE tumor samples stored in biobanks can now be analyzed.

In a pilot study we have been able to obtain allele-specific copy number information from 37 out of 52 ovarian cancer samples. These samples have been difficult to analyze with conventional methods (CN info from 10 out of 52 samples). Thus, we think that this technology will be important to obtain results from large cohorts also of relatively rare tumors. So if you are interested in studying the genomes of tumors in FFPE samples, get in touch with us and we will be able to tell you more.

 - CytoScan HD Cytogenetics Solution

The CytoScan HD Solution provides the broadest coverage, highest performance, and most "genotype–able" single nucleotide polymorphisms (SNPs) for detecting human chromosomal aberrations associated with genes related to constitutional and cancer cytogenetics, OMIM, and RefSeq. This single array includes more than 2.6 million copy number markers of which 750,000 are "genotype–able" SNPs and 1.9 million are non–polymorphic probes.

Bioinformatiksupport

We offer all types of services from the initial consultation, performing the complete array experiments to analyzing the results.

Since we are focusing on using the commercially available Affymetrix technology we provide all types of analysis for the applications available. We have many years’ experience  of expression analysis, copy number analysis of tumor samples and methylation data analysis so we can offer service and support on all steps of the analysis procedure here including experimental design, quality control, normalization, visualization, various statistics and evaluation of the results and integrating different data types.

For more information contact:

Amrei Binzer:
amrei.binzer[at]medsci.uu.se | 018-4714484

Pascal Pucholt:
pascal.pucholt[at]medsci.uu.se | 018-4714484

General bioinformatics questions can also be discussed on  www.biosupport.se.

Protokoll och provberedning

Affymetrix protocols

Expression analysis

 - miRNA

 - Whole transcriptome analysis - Gene ST and XTA arrays

- WT Plus Reagent Kit
- WT Plus Reagent Kit Assay Manual

- 3' Gene Expression arrays

- 3' IVT Plus Kit
- 3' IVT PLUS Reagent Kit Assay Manual


Genotyping

- Axiom Genotyping Solution

- Axiom Genotyping Assay
- Axiom Genotyping Assay Manual


Copy number analysis

- Cytoscan HD Assay

- Cytoscan HD Assay product information
- Cytoscan HD Assay Manual

- Oncoscan FFPE Assay

- Oncoscan FFPE Assay product information
- Oncoscan FFPE Assay Manual

- Genome Wide Human SNP Array 6.0

- please inquire


Sample Preparation Information

Method - our starting amount (recommended starting amount)

3' IVT express - 250 ng (50-250 ng)
Gene ST or XTA - 250 ng (100-300 ng )
Cytoscan HD - 250 ng (250 ng)
Mapping 6.0 - 500 ng (500 ng)
Regarding concentrations for Mapping assays we would like 50 ng/uL, but at least 20 ng/uL is necessary.
For Axiom or Oncoscan please inquire.


Extraction methods:

RNA extraction

- using Trizol(requires further purification, eg. Qiagen RNeasy)
- using Qiagen (or any other similar method)

DNA extraction

- Qiagen protocol (or any other similar method)

Forskning och utveckling

Our aim is to develop and apply bioinformatic tools to describe and understand biological processes involved in human disease so that improved treatments can be developed. We focus on how to use large-scale molecular data including microarray data to obtain these goals. In practice it means that we are involved in two types of research acivities.

1) Bioinformatic tools

We develop improved bioinformatic tools that can provide better answers to relevant research questions.

Our efforts to develop new bioinformatic tools have been focused on three major areas over the last few years: genomic aberrations in tumor cells, non-coding RNAs from RNAseq data and improved performance estimation.

Identification of genomic aberrations in tumor cells

The goal is to identify copy number aberrations in tumor cells in clinical tumor samples. However there are a number of issues that makes this task difficult. Often large proportions of non-tumor cells are present in the sample. Tumor cells are often aneuploid complicating normalization. Different tumor cells may also have different aberrations.

Whole genome sequencing data

We have developed a bioinformatic tool called Patchwork that uses whole-genome sequencing data from cancer samples to obtain segments with allele-specific copy numbers.

Mayrhofer M, DiLorenzo S, Isaksson A. 2013. Patchwork: allele-specific copy number analysis of whole genome sequenced tumor tissue. Genome Biol. Mar 25;14(3):R24

SNP array data

We have also developed one bioinformatic tool called Tumor Aberration Prediction Suite (TAPS) identifies allele-specific copy numbers and loss-of heteozygosity from SNP array data, even in the presence of large proportions of non-tumor cells and aneuploidy.

2) Data analysis

We apply publically available tools or those we have developed ourselves to perform data analysis for particular biomedical applications.

Our efforts to analyze data in biomedical applications fall into two broad categories: Analysis of genomic DNA, mRNA expression and/or methylation in tumor samples and Identification of mRNA biomarkers in various types of samples

Analysis of genomic DNA, mRNA expression and/or methylation in tumor samples

Kanduri M, Marincevic M, Halldórsdóttir AM, Mansouri L, Juvenik K, Ntoufa S, Göransson Kultima H, Isaksson A, Juliusson G, Andersson P-O, Ehrencrona H, Stamatopoulos K, Rosenquist R. 2012. Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles. Epigenetics Dec 1; 7(12):1435-42

Sooman, L.; Ekman, S.; Andersson, C.; Johansson, F.; Goransson-Kultima, H.; Isaksson, A.; Bergqvist, M.; Blomquist, E.; Lennartsson, J.; Gullbo, J. 2012. 1012 Synergistic Effects of PI3K or P38 MAPK Inhibition in Combination With Vandetanib Treatment in Glioblastoma Cells. European Journal of Cancer vol. 48 July, 2012. p. S244

Skirnisdottir I, Mayrhofer M, Rydåker M, Åkerud H and Isaksson A. 2012.Loss-of-heterozygosity on chromosome 19q in early-stage serous ovarian cancer is associated with recurrent disease. BMC Cancer2012, 12:407doi:10.1186/1471-2407-12-407

Cahill N, Bergh AC, Kanduri M, Göransson-Kultima H, Mansouri L, Isaksson A, Ryan F, Smedby KE, Juliusson G, Sundström C, Rosén A, Rosenquist R. 2012. 450K-array analysis of chronic lymphocytic leukemia cells reveals global DNA methylation to be relatively stable over time and similar in resting and proliferative compartments. Leukemia. 2013 Jan;27(1):150-8. doi: 10.1038/leu.2012.245. Epub 2012 Aug 27

Mansouri L, Gunnarsson R, Sutton LA, Ameur A, Hooper SD, Mayrhofer M, Juliusson G, Isaksson A, Gyllensten U, Rosenquist R. 2012. Next generation RNA-sequencing in prognostic subsets of chronic lymphocytic leukemia. Am J Hematol. 2012 Jul;87(7):737-40.

Edlund K, Lindskog C, Saito A, Berglund A, Pontén F, Göransson-Kultima H, Isaksson A, Jirström K, Planck M, Johansson L, Lambe M, Holmberg L, Nyberg F, Ekman S, Bergqvist M, Landelius P, Lamberg K, Botling J, Ostman A, Micke P. 2012. CD99 is a novel prognostic stromal marker in non-small cell lung cancer. Int J Cancer. 2012 131(10):2264-73

Halldórsdóttir AM, Kanduri M, Marincevic M, Mansouri L, Isaksson A, Göransson H, Axelsson T, Agarwal P, Jernberg-Wiklund H, Stamatopoulos K, Sander B, Ehrencrona H, Rosenquist R. 2012. Mantle cell lymphoma displays a homogenous methylation profile: A comparative analysis with chronic lymphocytic leukemia. Am J Hematol 2012 Apr;87(4):361-7. doi: 10.1002/ajh.23115. Epub 2012 Feb 28.

Schmidt M, Hellwig B, Hammad S, Othman A, Lohr M, Chen Z, Böhm D, Gebhard S, Petry IB, Lebrecht A, Cadenas C, Marchan R, Stewart J, Solbach C, Holmberg L, Edlund K, Kultima HG, Rody A, Berglund A, Lambe M, Isaksson A, Botling J, Karn T, Müller V, Gerhold-Ay A, Cotarelo C, Sebastian M, Kronenwett R, Bojar H, Lehr HA, Sahin U, Koelbl H, Gehrmann M, Micke P, Rahnenführer J, Hengstler JG. 2012. A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin kappa C as a compatible prognostic marker in human solid tumors. Clin Cancer Res. 2012 May 1;18(9):2695-703. doi: 10.1158/1078-0432.CCR-11-2210. Epub 2012 Feb 20.

Micke P, Edlund K, Holmberg L, Kultima HG, Mansouri L, Ekman S, Bergqvist M, Scheibenflug L, Lamberg K, Myrdal G, Berglund A, Andersson A, Lambe M, Nyberg F, Thomas A, Isaksson A, Botling J. Gene copy number aberrations are associated with survival in histologic subgroups of non-small cell lung cancer. J Thorac Oncol. 2011 Nov;6(11):1833-40.

Gunnarsson R, Mansouri L, Isaksson A, Göransson H, Cahill N, Jansson M, Rasmussen M, Lundin J, Norin S, Buhl AM, Smedby KE, Hjalgrim H, Karlsson K, Jurlander J, Geisler C, Juliusson G, Rosenquist R. Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia. Haematologica. 2011 Aug;96(8):1161-9. Epub 2011 May 5.

Halldórsdóttir AM, Sander B, Göransson H, Isaksson A, Kimby E, Mansouri M, Rosenquist R, Ehrencrona H. (2011) High-resolution genomic screening in mantle cell lymphoma-specific changes correlates with genoic complexity, the proliferative signature and survival. Genes Chromosomes Cancer. 2011 Feb;50(2):113-21.

Marincevic M, Mansouri M, Kanduri M, Isaksson A, Goransson H, Ekstrom Smedby K, Jurlander J, Juliusson G, Davi F, Stamatopoulos K, Rosenquist R. 2010. Distinct gene expression profiles in subsets of chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B cell receptors. Haematologica. 2010 Aug 26.

Marincevic M, Cahill M, Gunnarsson R, Isaksson A, Mansouri M, Göransson H, Rasmussen M, Jansson M, Ryan F, Karlsson K, Adami HO, Davi F, Jurlander J, Juliusson G, Stamatopoulos K and Rosenquist R. 2010. High-density screening reveals diferent sepctra of genomic aberrations in chronic lymphosytic leukemia patients with “stereotyped” IGHV3-21 or IGHV4-34 B-cell receptors. Haematologica. Apr 26.

Kanduri M, Cahill N, Göransson H, Enström C, Ryan F, Isaksson A and Rosenquist R. 2010. Differential genome-wide methylation profiles in prognostic subsets of chronic lymphocytic leukemia. Blood. Jan 14;115(2):296-305

Gunnarsson R, Isaksson A, Mansouri M, Göransson H, Jansson M, Cahill N, Rasmussen M, Staaf J, Lundin J, Norin, Buhl AM, Ekström Smedby K, Hjalgrim H, Karlsson K, Jurlander J, Juliusson G and Rosenquist R. 2010. Large but not small copy-number alterations correlate to high-risk genomic aberrations and survival in chronic lymphocytic leukemia: A high-resolution genomic screening of newly diagnosed patients. Leukemia. Jan;24(1):211-5

Gunnarsson R, Staaf J, Jansson M, Ottesen AM, Göransson H, Liljedahl U, Ralfkiaer U, Mansouri M, Buhl AM, Ekström Smedby K, Hjalgrim H, Syvänen A-C, Borg Å, Isaksson A, Jurlander J, Juliusson G and Rosenquist R. 2008. Screening for copy number alterations and loss-of-heterozygosity in Chronic Lymphocytic Leukemia -A comparative study of four differently designed high-resolution microarray platforms. Genes Chromosomes and Cancer 2008 Aug;47(8):697-711

Fryknäs M, Dhar S, Öberg F, Rickardson L, Rydåker M, Göransson H, Gustafsson MG, Pettersson U, Nygren P, Larsson R and Isaksson A. 2007. STAT1 signaling is associated with acquired cross-resistance to doxorubicin and radiation in myeloma cell lines. Int J Cancer. Jan 1;120(1):189-95.
 

Identification of mRNA biomarkers in various types of samples

Brunberg E, Jensen P,Isaksson A, Keeling L. 2012. Brain gene expression differences are associated with abnormal tail biting behavior in pigs. Genes Brain Behav. 2012 Nov 12. doi: 10.1111/gbb.12002.

Zhao H, Dahlö M, Isaksson A, Syvänen AC, Pettersson U. The transcriptome of the adenovirus infected cell. Virology. 2012 Jan 9.

Ronquist GK, Larsson A, Ronquist G, Isaksson A, Hreinsson J, Carlsson L, Stavreus-Evers A. Prostasomal DNA characterization and transfer into human sperm. Mol Reprod Dev. 2011 Jul;78(7):467-76. doi: 10.1002/mrd.21327. Epub 2011 Jun 2.

Brunberg E, Jensen P, Isaksson A, Keeling L. Feather pecking behavior in laying hens: hypothalamic gene expression in birds performing and receiving pecks.Poult Sci. 2011 Jun;90(6):1145-52.

Laryea D, Gullbo J, Isaksson A, Larsson R, Nygren P. 2010. Characterization of the cytotoxic properties of the benzimidazole fungicides, benomyl and carbendazim in human tumor cell lines and primary cultures of patient tumor cells. Anticancer Drugs. 2010 Jan;21(1):33-42