University of the basque country UPV/EHUScience and technology faculty Edificio CD4Maria Goyri research building, CBA at UPV/EHU science parkBº Sarriena, s/n 48940 Leioa (Vizcaya)
Dr. Irantzu Bernales Phone: 94 601 3497 e-mail: firstname.lastname@example.org
Access to the unit of Gene Expression involves meeting the requirements set forth in the Protocol for access to SGIker and the services it provides. Check website for further details.
One of the most surprising and incredible phenomena in biology is the accuracy with which the correct expression is coordinated for thousands of genes from an organism: each gene must be expressed at a suitable moment in time and place to ensure proper cellular function. In terms of establishing the function of the genes, knowing when, where and how they are expressed is essential for understanding the activity and biological role of the proteins encoded by these genes. Furthermore, changes in gene expression patterns may provide indications about regulating mechanisms and also cellular functions and metabolic routes. On the other hand, awareness of the regulation of the gene expression and gene interactions which are formed in each cellular type for full knowledge about the molecular causes of diverse complex diseases such as cancer, neuro-degenerative diseases and ageing processes. Knowledge about the molecular causes of such pathologies would entail the development of new methods of diagnosis, and new therapeutic products used for treatment. Functional genomics and, specifically, microarray technology, enables the genes associated with physiological and pathological processes to be identified. Information deriving from such identification enables new early research systems to be developed and the genetic predisposition towards being affected by a disease to be assessed, as well as enabling new drugs to be developed that are able to combat illnesses which are difficult to cure at present. Large-scale gene expression analysis via the use of high-density microarray technology using oligonucleotides or additional DNAs enables biological problems to be tackled from a global standpoint, and has brought about a major revolution in the study of normal and pathological gene regulation in areas such as human, animal, plant and microbial biology. Although the most important application of microarrays involves monitoring global gene expression, they are also used in the study of gene regulation by analysing the joining of proteins to promoter sequences of genome genes as a whole (ChIP-chip microarrays), and in the study of delection or expansion of genomic regions (aCGH microarrays) - a very common type of alteration in cancer. The Gene Expression Unit of the Genomics and Proteomics Service at the University of the Basque Country, UPV/EHU, has been set up in order to provide full back-up on both an experimental and intellectual level to researchers belonging to the Basque Network of Science, Technology and Innovation, to biocompanies from the milieu, and to public and private institutions throughout Spain and overseas, in activities related to the study of genes and genomes. To this end, it has qualified personnel at its disposal together with state-of-the-art, high-performance equipment and a recently-created laboratory that has been designed in accordance with the recommendations of laboratory good practices. The Gene Expression Unit offers the following systems for comparative analysis of gene expression, in addition to other applications: Quantitative or real-time PCR, using the 7900HT Fast Real Time PCR system made by Applied Biosystems. Measurement of the gene expression using real-time or quantitative PCR (qPCR) is based on the detection of fluorescence by a fluorochrome reporter, whose signal increases proportionally to the amount of product generated in each PCR cycle. This technique permits more accurate quantitative analyses which are sensitive to nucleic acids. Two types of chemistry are used to detect the PCR product: specific fluorescent colouring for SYBR Green double-stranded DNA, or specific fluorescent probes known as TaqMan. Microarray Platform made by Agilent Technologies, one of the most advanced microarray systems providing a high quality and very reproducible methodology. Agilent microarrays are made up of specific 60-mer oligonucleotides arranged on a matrix with high sensitivity, which enables low amounts of initial RNA to be used. Agilent at present offers a series of microarrays that haven been pre-designed for applications such as monitoring gene expression, ChIP-chip or CGH (aCGH) arrays, although it also enables tailor-made microarrays to be designed with the probes chosen by the customer for specific studies in the field being developed by their research. RNA quality control analysis using the Agilent 2100 Bioanalyzer.
The Gene Expression Unit of the Genomics and Proteomics Service offers scientific advice and technical back-up, and provides the basic infrastructure to enable researchers from the University of the Basque Country and other public and private institutions from the Basque Autonomous Community and neighbouring regions to incorporate microarray technology and gene expression analysis into their projects. The services offered by the Unit include the experiments and protocols required in all projects involving the application of microarray technology, ranging from the experimental design of microarray experiment, processing and quality control of the samples, microarray processing and analysis of results, to the validation of those results and gene expression analysis using real-time PCR. The Unit also offers training courses that mainly cover protocols and analysis of results. Specifically, the Unit offers the following services:
- RNA extraction from cells and animal tissues. Please check with the Unit for any other type of tissue or bacteria.
- Quality control (QC) of RNA samples for use in the high-density microarray system and real-time PCR. (See the document)
- Microarray service provided by the Agilent Technologies platform. (See the document)
- Advice on experimental design and analysis of results.
- Advice and design of tailor-made arrays.
- Complete processing of Agilent Technologies microarrays of different formats: High Definition (HD) microarrayak (1x244K, 2x105K, 4x44K, 8x15K); SurePrint G3 microarrayak (1x1M, 2x400K, 4x180K, 8x60K).
- Complete processing of Gene Expression, Exon, ChIP-on-chip, comparative genomic hybridization and Copy Nunmber Variation (CGH, CNV, CGH + SNP), and microRNA (miRNA) microarrays.
- Gene Expression analysis using real-time PCR or quantitative PCR (Q-PCR). (See the document)
- Advice on experimental design.
- Design of primers and TaqMan probes using the PrimerExpress software (Applied Biosystems).
- cDNA synthesis.
- Optimization of primers for SYBR Green.
- Development of real-time PCR with 3 block format options: 96-Fast, 384-well and TLDA or MFC blocks, using two chemical techniques: SYBR Green or specific TaqMan probes.
- High Throughput gene expression and absolute quantification projects with the nanofluidic BioMark HD real time PCR System (Fludigm Corporation) 48.48 and 96.96 Dynamic Arrays for gene expression, and Digital Arrays for absolute quantification.
- Data analysis.
Some of the equipment is shared with the Genomics and Proteomics Sequencing and Genotyping Service.
Equipped with a NanoLockSpray system and connected to a HPLC Waters CapLC XE system and MassLynx computer platform.
The features of the equipment are as follows:
With MALDi and ESI ionization sources and Q-Exactive spectrometer.