Genómica funcional

Herbert Auer

Jefe de Genómica Funcional

Tel Oficina : +34 93 403 98 03

correo-e : herbert.auerirbbarcelona.org

Introducción

The IRB Barcelona Functional Genomics Core Facility (FGC) serves researchers from academia and industry. We provide comprehensive support to projects whenever genome-wide measurements of DNA and RNA, and the manipulation of gene expression are required.

Tools for Genomic Analysis

The facility provides tools for:

• miRNAs
• transcriptomics
• DNA copy numbers
• ChIP-Seq
• genomic sequencing
• SNPs

Genome-wide measurements of DNA alterations and gene expression on the RNA level can be a challenge during research projects. The facility provides a complete service to work with researchers throughout the entire project. In addition to cutting-edge tools such as microarrays from Affymetrix and NimbleGen and Next Generation Sequencing by Illumina, the facility provides a consultancy service regarding genomic analysis.

Alteration of Gene Expression

The FGC provides IRB Barcelona and IBMB (CSIC) researchers with the following clones:

• human shRNAs
• mouse shRNAs
• human ORFs (for IRB Barcelona only)

Reverse genetics calls for efficient tools to alter gene expression. The FGC provides almost 200,000 clones for genetic manipulation. These include all of Sigma's TRC1 human and mouse shRNA clones for knock-down of expression, together with Open Biosystems human ORF clones for over-expression. ORF clones are also useful as probes for in situ hybridisation or Northern blots.

shRNA clones come in lentiviral vectors for efficient infection of many cell types, including non-dividing ones.

ORF clones contain the gateway system for hassle-free transfer into any kind of expression vector.

Más información

• 2008 Scientific Report - Functional Genomics Core Facility
• 2007 Scientific Report - Functional Genomics Core Facility

Genómica funcional

Tools for genomic analysis

Determining the type of genomic analysis tools that work best for a specific question depends on the nature of the question. In general, questions regarding known and well-characterised transcripts (mRNAs and miRNAs) are answered more straight forward using microarrays. Gene discovery and characterisation of ChIP material is done more efficiently by sequencing. Some frequently interrogated themes are described here:

RNA

microRNA analysis
The FGC provides the Affymetrix miRNA GeneChip for miRNA analysis. For over 70 organisms, a comprehensive analysis of known miRNAs is performed on a single array. Minimal amount: 250 ng total RNA

Discovery of novel small RNAs (miRNAs, piRNAs and other types of small RNAs) is most efficiently performed by sequencing. Minimal amount: 1000 ng total RNA

mRNA expression profiling
For over 20 organisms, we provide Affymetrix expression arrays for mRNA profiling. These arrays provide a robust and reliable tool to quantify expression of the majority of known genes. Minimal amount: 50 ng total RNA for ST arrays, 5 ng for 3' arrays

For over 100 organisms, we provide NimbleGen expression arrays for mRNA profiling. These arrays are especially suited for small genomes since multiple samples can be analysed on a single array, thereby leading to cost reduction. Minimal amount: 1000 ng total RNA

RNA Seq
By nature, this is performed by sequencing. It allows the characterisation of alternative splicing, transcriptional start and stop sites and the discovery of novel transcripts. Sample processing and data analysis are highly experimental and do not follow standard operating procedures yet. Minimal amount: 5000 ng total RNA

DNA

DNA copy number variation (CNV analysis)
CNV analysis at a resolution of 10-100kb is performed on all organisms for which expression arrays are available from Affymetrix or NimbleGen. In addition, NimbleGen provides a broad spectrum of arrays dedicated to CNV analysis for many organisms. Minimal amount: 5000 ng genomic DNA

CNV analysis is also performed by genome sequencing, especially for organisms with a smaller genome (Drosophila, yeast etc.). Together with CNV information, many other types of genetic alterations (point mutations, InDels, translocations, inversions) are studied in the same experiment. Minimal amount: 5000 ng genomic DNA

ChIP-Seq
Characterisation of transcription factor binding sites and location of histone modifications is best performed by sequencing ChIP material. In organisms with a complex genome like the human or mouse, the optimal baseline (control antibodies, ChIP-input) has not yet been determined. Minimal amount: 10 ng ChIPed DNA

DNA methylation
Analysis of multiple loci for DNA methylation is still performed in an experimental phase. For specific projects regarding DNA methylation, please contact us.

Alteration of gene expression

Reverse genetics calls for efficient tools to alter gene expression. The FGC provides IRB Barcelona and IBMB (CSIC) researchers with almost 200,000 clones for genetic manipulation. These include all Sigma's TRC1 shRNA clones for human and mouse for knock-down of expression and Open Biosystems' human ORF clones for over-expression (IRB Barcelona users only). ORF clones are also useful as probes for in situ hybridization or Northern blots.

Human & mouse shRNAs
shRNA clones come in lentiviral vectors for efficient infection of many cell types, including non-dividing ones. On average, we provide our internal users with 4-5 different clones per gene. In many cases, the clones targeting the same gene provide different knock-down efficiencies and help to control off-target effects. Approximately 75% of all human and mouse refseq genes are targeted. The complete list of shRNA clones is available on www.dnaarrays.org. Detailed information about the TRC1 clones is available on Sigma Aldrich’s website.

Human ORF clones
The Open Biosystems ORF library provides IRB Barcelona researchers with approximately 13,000 clones for expression of one ORF per gene. These clones are most frequently used for over-expression of genes but can also be used as probes. ORF clones contain the Gateway system for hassle-free transfer into any kind of expression vector.

The list of available ORF clones can be consulted on www.dnaarrays.org. Detailed information about the ORF clones is available on Open Biosystem's ORF webpage.

Genómica funcional

General

Initial consultation
Prior to starting your experiments, we invite you to discuss the experimental design with us.

Requirements for the services
Every service (clone delivery, microarray analysis, sequencing...) has its own specifications; details are available on the request forms of the specific services. Request forms can be downloaded from our website www.dnaarrays.org.

Who can perform projects with the FGC?
The FGC facility serves both internal and external researchers. We work with academia as well as with industry.

How is payment of services organised at the FGC?
In general we have two fees, one applicable for IRB Barcelona researchers and the other for external entities. Our pricing policy is very transparent: Download our request forms, type in the number of samples you wish to process on a certain platform and the price is shown automatically. For Spanish researchers: The government still does not give research institutions a tax-exemption status. Therefore, VAT (IVA) must be added to the prices shown.

Tools for Genomic Analysis

How long does it take until I get my results?
For smaller microarray projects (<24 samples), you can expect to receive your data 3 weeks after sample and order form delivery. For special arrays, Affymetrix and NimbleGen can take longer for array delivery.

For sequencing projects, no specific time frame can be given for several reasons: Illumina does not store consumables in Europe - therefore, and for other Illumina-internal reasons, our orders may be delayed. Please also be aware that one sequencer run processes 8 samples in parallel. All of them have to be the same read-length and either single-end or paired-end reads. The complete flow cell needs to be filled to provide the price indicated in our request forms. This implies either that you fill the entire flow cell with your samples or you wait until others wish to do something similar.

Which organisms can be analysed?
Any organisms with a well-annotated genome (or transcriptome) can be analysed. In the field of microarrays, standard arrays can be purchased for many organisms. Affymetrix covers over 20 organisms, NimbleGen over 100. Arrays can also be customised. This is usually easier with NimbleGen than with Affymetrix. In the field of Next Generation Sequencing, a reference genome of the organism must be available. Sequences are mapped to this reference genome.

How do I best prepare my samples?
In general, a combination of organic phase extraction (phenol) with column purification (e.g. Qiagen) works best. These two completely different purification principles remove all kind of impurities. Protocols can be found on our website. For miRNA analysis do not use columns since almost all of them remove short RNAs. For very small samples (a few thousand cells), specific protocols are necessary, and these can also be found on our website www.dnaarrays.org.

I have doubts about the quality of my samples. Should I still submit them for analysis?
Before we start an expensive experiment like arrays or sequencing, we perform a broad range of quality controls. These include Nanodrop testing of purity, RNA integrity test on the Bioanalyser, and DNA or RNA specific quantification using the Qubit assays. If we have doubts about the samples, we will contact you.

Alteration of Gene Expression

Who can request clones?
IRB Barcelona has a single-use contract with Sigma and Open Biosystems, i.e. only IRB Barcelona and IBMB-CSIC researchers have access to the Sigma libraries while only IRB Barcelona researchers have access to the latter. If you work for another organisation please contact Sigma and/or Open Biosystems in order to access these clones.

For which genes do you have shRNA or ORF clones?
Our shRNA clones cover approximately 75% of human and mouse refseq mRNAs, the ORF clones cover the same percentage of human genes. Complete lists of clones available and corresponding genes can be found on our website.

I cannot open the clone lists because they are in xlsx-format
The libraries contain more than 65,535 clones. Therefore, Excel 2007 is required to show more than 65,535 rows.

I cannot find my favourite gene in your clone lists
Our lists contain the official gene names as available on the NCBI website. At NCBI, go to the search field, click on gene and check if you have used the official name.

How long does it take until I receive my clones?
Send us an email with your clone request and we will inform you when tubes for bacterial inoculation should be delivered. You will receive the clones the same day as tube delivery. Process takes a maximum of three days.

Genómica funcional

Manager
Herbert Auer
tel +34 93 403 98 03
e-mail: herbert.auerirbbarcelona.org

Senior Research Officers
Silvia Rodríguez Mulero
tel +34 93 403 98 03
e-mail: silvia.rodriguezirbbarcelona.org

Annie Rodolosse
tel +34 93 403 98 03
e-mail: annie.rodolosseirbbarcelona.org

Genómica funcional

The DNA-Proteome: Recent advances towards establishing the protein-DNA interaction space
Grotewold E and Auer H.
Int J Comput Bioscience, 1, 1008 (2010)

Expression Profiling Using Affymetrix GeneChip Microarrays
Auer H, Newsom DL and Kornacker K.
Methods Mol Biol, 509, 35-46 (2009)

Autism-specific copy number variants further implicate the phosphatidylinositol signaling pathway and the glutamatergic synapse in the etiology of the disorder
Cuscó I, Medrano A, Gener B, Vilardell M, Gallastegui F, Villa O, González E, Rodríguez-Santiago B, Vilella E, Del Campo M and Pérez-Jurado LA.
Hum Mol Genet, 18, 1795-1804 (2009)

Innate and adaptive immune activation in the brain of MPS IIIB mouse model
DiRosario J, Divers E, Wang C, Etter J, Charrier A, Jukkola P, Auer H, Best V, Newsom DL, McCarty DM and Fu H.
J Neurosci Res, 87 (4), 978-990 (2009)

Expression divergence and copy number variation in the human genome
Auer H.
Cytogenet Genome Res, 123 (1-4), 278-282 (2008)

Islet graft response to transplantation injury includes upregulation of protective as well as apoptotic genes
Rodríguez-Mulero S and Montanya E.
Cell Transplant, 17 (9), 1025-1034 (2008)

Array-CGH in patients with Kabuki-like phenotype: identification of two patients with complex rearrangements including 2q37 deletions and no other recurrent aberration
M, Vilardell M, González E, Gener B, Galán E, Toledo L and Pérez-Jurado LA.
BMC Med Genet, 11 (27) (2008)

Analysis of cytogenetic abnormalities in squamous cell carcinoma by array comparative genomic hybridization
Salgado R, Toll A, Espinet B, González-Roca E, Barranco CL, Serrano S, Solé F and Pujol RM.
Actas Dermosifiliogr, 99 (3), 199-206 (2008)

Transcriptional profiling of the megabladder mouse: a unique model of bladder dysmorphogenesis
Singh S, Robinson M, Ismail I, Saha M, Auer H, Kornacker K, Robinson ML, Bates CM and McHugh KM.
Dev Dyn, 273 (1), 170-186 (2008)

HOXB4's road map to stem cell expansion
Schiedlmeier B, Santos AC, Ribeiro A, Moncaut N, Lesinski D, Auer H, Kornacker K, Ostertag W, Baum C, Mallo M and Klump H.
Proc Natl Acad Sci USA, 104, 16952-16957 (2007)

Disruption of ZAS3 in mice alters NF-kappaB and AP-1 DNA binding and T-cell development
Allen CE, Richards J, Muthusamy N, Auer H, Liu Y, Robinson ML, Barnard JA and Wu LC.
Gene Expr, 14 (2), 83-100 (2007)

Big results from small samples: evaluation of amplification protocols for gene expression profiling
Viale A, Li J, Tiesman J, Hester S, Massimi A, Griffin C, Grills G, Khitrov G, Lilley K, Knudtson K, Ward B, Kornacker K, Chu CY, Auer H and Brooks AI.
J Biomol Tech, 18 (3), 150-161 (2007)

Gene-resolution analysis of DNA copy number variation using oligonucleotide expression microarrays
Auer H, Newsom DL, Nowak NJ, McHugh KM, Singh S, Yu CY, Yang Y, Wenger GD, Gastier-Foster JM and Kornacker K.
BMC Genomics, 8, 111 (2007)

Gene expression and functional evidence of epithelial-to-mesenchymal transition in papillary thyroid carcinoma invasion
Vasko V, Espinosa AV, Scouten W, He H, Auer H, Liyanarachchi S, Larin A, Savchenko V, Francis GL, de la Chapelle A, Saji M and Ringel MD.
Proc Natl Acad Sci USA, 104 (8), 2803-2808 (2007)

Quantification of DNA methylation in electrofluidics chips (Bio-COBRA)
Brena RM, Auer H, Kornacker K and Plass C.
Nat Protoc, 1 (1), 52-58 (2006)

ADOA3R as a therapeutic target in experimental colitis: proof by validated high-density oligonucleotide microarray analysis
Guzman J, Yu JG, Suntres Z, Bozarov A, Cooke H, Javed N, Auer H, Palatini J, Hassanain HH, Cardounel AJ, Javed A, Grants I, Wunderlich JE and Christofi FL.
Inflamm Bowel Dis, 12 (8), 766-789 (2006)

20q11.1 amplification in giant-cell tumor of bone: Array CGH, FISH, and association with outcome
Smith LT, Mayerson J, Nowak NJ, Suster D, Mohammed N, Long S, Auer H, Jones S, McKeegan C, Young G, Bos G, Plass C and Morrison C.
Genes Chromosomes Cancer, 45 (10), 957-966 (2006)

The ABRF MARG microarray survey 2005: taking the pulse of the microarray field
Knudtson KL, Auer H, Brooks AI, Griffin C, Grills G, Hester S, Khitrov G, Lilley KS, Massimi A, Tiesman JP and Viale A.
J Biomol Tech, 17 (2), 176-186 (2006)

Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform
Brena RM, Auer H, Kornacker K, Hackanson B, Raval A, Byrd JC and Plass C.
Nucleic Acids Res, 34 (3), e17 (2006)

MYC amplification and polysomy 8 in chondrosarcoma: array comparative genomic hybridization, fluorescent in situ hybridization, and association with outcome
Morrison C, Radmacher M, Mohammed N, Suster D, Auer H, Jones S, Riggenbach J, Kelbick N, Bos G and Mayerson J.
J Clin Oncol, 23 (26), 9369-9376 (2005)

Role of cancer-associated stromal fibroblasts in metastatic colon cancer to the liver and their expression profiles
Nakagawa H, Liyanarachchi S, Davuluri RV, Auer H, Martin EW Jr, de la Chapelle A and Frankel WL.
Oncogene, 23 (44), 7366-7377 (2004)

Acute myeloid leukemia with complex karyotypes and abnormal chromosome 21: Amplification discloses overexpression of APP, ETS2, and ERG genes
Baldus CD, Liyanarachchi S, Mrozek K, Auer H, Tanner SM, Guimond M, Ruppert AS, Mohamed N, Davuluri RV, Caligiuri MA, Bloomfield CD and de la Chapelle A.
Proc Natl Acad Sci USA, 101 (11), 3915-3920 (2004)

Tissue-wide expression profiling using cDNA Subtraction and Microarrays to identify tumor-specific genes
Amatschek S, Koenig U, Auer H, Steinlein P, Pacher M, Gruenfelder A, Dekan G, Vogl S, Kubista E, Heider KH, Stratowa C, Schreiber M and Sommergruber W.
Cancer Res, 64 (3), 844-856 (2004)

Genómica funcional

• 25 Febrero 2010
  International Journal of Computational Bioscience
  El Proteoma del ADN