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Affymetrix GeneChip system
The Genomics Platform is equipped with the latest version of the Affymetrix GeneChip system.
Affymetrix offers a complete solution for multiple DNA- and RNA-based applications:
• gene expression
• SNP genotyping
• unbiased transcriptome profiling
• analysis of protein-DNA interactions (ChIP on chip)
• resequensing
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Gene expression
For standard RNA-based applications (gene expression) Affymetrix proposes microarrays (GeneChips) covering numerous species, including human, mouse, rat, as well as numerous additional widely used model organisms (details on Affymetrix website).
These GeneChips contain probes designed toward the 3’ of transcripts. They enable evaluating gene expression using 3’ based assays that rely on oligo dT based amplification of cellular mRNA.
Recently, Affymetrix has introduced exon and gene arrays for human, mouse and rat (details on Affymetrix website).
These arrays contain probes covering multiple exons (for Gene arrays) or every exons (for exons GeneChips) with the aim of increasing sensitivity and enable to capture alternative splicing events.
The assays for target preparation for these arrays are based on a random priming strategy (with an optional ribosomal RNA depletion step for increased sensitivity before target synthesis).
Gene expression or global genome organization
Major improvements in GeneChips manufacturing now allow Affymetrix to produce GeneChips containing more than 6 millions features.
As a result, tiling arrays for human, mouse, rat and some additional model organisms are now available.
These arrays contain probes regularly spaced along chromosomal sequences, irrespective of genome annotation (repetitive sequences being excluded).
Tiling arrays permit new analysis of the genome, such as unbiased transcriptome analysis, genome-wide analysis of chromatin immunoprecipitation (ChIP on chip) (see Products and Services on Affymetrix website).
Genotyping
For genotyping Affymetrix proposes a range of GeneChips with various numbers of SNPs interrogated, depending on the requirements of the projects.
Target preparation from genomics DNA involves enzymatic digestion followed by PCR amplification under conditions that reduce genome complexity.
Fragmented and labeled DNA is subsequently hybridized onto the GeneChip. Genotyping products comprise GeneChips covering up to 906,600 SNPs and containing more than 946,000 probes for CNVs
(see GeneChip arrays on Affymetrix website).
The Platform covers these applications. ›› Services
The Platform is equipped with 2 complete GeneChip systems including each a hybridization oven, two Fluidics 450 washing/staining stations, and a 7G scanner.
Illumina BeadStation
The Platform is equipped with one Illumina’s BeadStation 500 system as well as an iScan instrument equipped with the Autoloader2
(see system overview on Illumina website).
This complete benchtop solution for genetic analysis enables a wide range of sample throughput and multiple applications including both RNA- and DNA-based applications:
• genome-wide or focused gene expression
• SNP genotyping
• LOH/copy number
• DNA methylation analysis
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Gene expression
For standard RNA-based applications (gene expression), Illumina proposes microarrays (BeadArrays) covering numerous species, including human, mouse, rat (details on Illumina website).
These BeadArrays contain probes designed toward the 3’ of transcripts.
They enable evaluating gene expression using 3’ based assays that rely on oligo dT based amplification of cellular mRNA.
The DASL assay enables to interrogate a restricted number of selected transcripts for large number of samples (details on Illumina website).
Genotyping
For genotyping, Illumina proposes a range of BeadArrays with various numbers of SNPs interrogated, depending on the requirements of the projects.
2 assays are available depending on the number of SNP to interrogate and the number of samples to process.
1. Infinium II assays for whole-genome. Genomics DNA is amplified using whole genome amplification assays and hybridized onto arrays containing oligonucleotides that terminate one base before the interrogated SNP.
SNP detection is performed by single base extension using dual colored assays (details on Illumina website).
Infinium products comprise BeadArrays covering up to 1 million SNPs and numerous CNVs.
2. Goldengate assays for targeted genotyping. gDNA is hybridized to SNP specific primers followed by multiplexed PCR amplification.
Amplified material is subsequently hybridized onto SAM.
Genotyping assays are robotized - on Tecan Freedom EVO instruments - for improved throughput and technical consistency.
| Real-time quantitative PCR |
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ABI PRISM SDS 7900 HT
We routinely use qRT PCR for the following major applications:
• validation of a subset of genes selected following a microarray experiment
• measure of low to moderate numbers of gene of interest
• genotyping (DNA copy number for transgenic animals and analysis of ploidy)
• analysis of chromatin immunoprecipitations
• measure of miRNA expression levels (TaqMan® technology from ABI; see technical sheet
- PDF; 6 pages, 886 KB)
Compared to microarrays, qRT PCR is much more precise and has a wider dynamic range.
Real-time PCR experiments are performed on our 7900HT SDS system from ABI (see system description on ABI website).
These instruments are equipped with automated devices for plates loading and are thus designed for high throughput, with high precision in measures and good flexibility.
qRT PCR is based on the measure of the accumulation of very short amplicons in real time.
We currently propose 2 alternative detection methods:
• Sybr Green: a non-specific dye which binds double-stranded DNA,
• TaqMan® probes: short oligonucleotides that are complementary to the amplicons.
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TaqMan® probes provide additional levels of specificity and enable more precise quantification for gene expression than Sybr Green.
The selection of the most appropriate detection method will depend upon the specific criteria of each project.
Additional applications with the ABI Prism SDS 7900 HT include the detection of single nucleotide polymorphisms (SNPs) using 5’ nuclease assays.
| nCounter (NanoString Technologies) |
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The nCounter from NanoString Technolo- gies is an instrument designed for highly multiplexed measures of gene expression using fluorescently labeled reporter probes.
Indeed through the use of a unique coding technology, direct counting of individual RNA molecules is made possible across all levels of biological expression, with sensitivity and specificity similar to Real Time PCR (RT PCR), and without any enzymatic reactions involved.
Applications include:
- mRNA profiling: custom set of genes or off-the-shelf preassembled panels (details on NanoString website);
- miRNA profiling: comprehensive collection of more than 700 human and human-associated viral miRNAs derived from miRBase (details on NanoString website).
The technology has been described in the following paper: Geiss et al., 2008. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnology 26, 317-325. (PDF; 12 pages, 4.32 MB)
The probes used are about 100 bases long. The system is thus very resistant to lower RNA quality and is perfectly suited for FFPE samples for example.
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The system includes build in controls.
- Positive controls: these include a series of 10 RNA spiked in the CodeSet and are used for internal QC and calibration.
- Negative controls: a series of 7 probes against alien sequences that are used for QC purposes and for generation of background values.
The complete system includes two instruments.
- PrepStation (liquid handling robot), used to purify ternary complexes obtained during the hybridization, remove unhybridized free probes and immobilize the complexes into the cartridge for subsequent image acquisition.
- nCounter (image acquisition instrument), used to scan the cartridge and generate raw data (counts).
For custom projects, probes for a CodeSet are designed by NanoString bioinformaticians, on the basis of a RefSeq ID’s. This format is available for 48 or 96 samples, or multiple of 96 samples.
Similar to RT PCR, probes for normalization genes must be included in the CodeSet. These must be selected based on prior knowledge.
For miRNA assays, the complete panel is provided in 12, 24, 48, and 96 sample sizes ( see a description of miRNA assay - PDF; 4 pages, 854 KB).
Data treatment proposed by the Platform includes background correction, normalization and fold-change calculation.
Benefits
• Low RNA input (100 ng of total RNA⁄sample)
• Possibility to analyze RNA samples of poor quality (such a FFPE samples)
• Up to 576 transcripts simultaneously interrogated per sample
• No enzymatic reactions involved
• Very high dynamic range, sensitivity and reproducibility
Applications
• Microarray validation
• Pathway analysis
• Expression of defined gene sets
• Biomarker validation
Service proposed by the Platform
• Initial discussion for experimental design
• Order and QC (BLAST) of the CodeSet
• Processing of the samples
• Data analysis
| Ultra high throughput sequencing |
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Illumina Genome Analyzer IIx
The Genomics Platform is equipped with a Genome Analyzer IIx (GA IIx) from Illumina.
This sequencing platform is based on massively parallel sequencing of millions of reads using proprietary reversible terminator-based sequencing chemistry (see method description on Illumina website).
The GA IIx is a highly robust, accurate and scalable system ideally suited for large scale sequencing applications.
The complete system includes a Cluster generation station, used to prepare DNA clusters for sequencing, and the GA IIx instrument for sequencing by synthesis.
Equipped with a paired-end module, the GA IIx enables sequencing of 38, 76 or 105 bases from one end (single read; SR) or both end (paired-end; PE) of DNA inserts.
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Library preparation can be performed by the Platform or the users. ›› Services
Applications (non exhaustive list)
• Whole-genome sequencing and resequencing
• Candidate region sequencing and resequencing
• mRNA sequencing (discovery, splicing variant analysis and gene expression)
• Small RNA (miRNA) discovery and analysis
• Analysis of chromatin immunoprecipitation (ChIP-seq)
• Analysis of methylation patterns
ABI PRISM 3100 Genetic Analyzer
The Genomics Platform is equipped with an ABI PRISM 3100 Genetic Analyzer ( see system description on ABI website).
This instrument is dedicated to the separation and sizing of DNA fragments by capillary electrophoresis, using the software GeneScan (ABI).
Note: the Genomics Platform is not a sequencing facility.
2100 Bioanalyzer (Agilent)
We routinely use the Agilent 2100 Bioanalyzer for quality control of the RNA samples before any application (see system description on Agilent website).
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SpectraMax Gemini XPS Fluorescence-Luminescence Reader
(Molecular Devices)
In order to improve the quality of our work on samples available in very low quantities
(e.g. LCM samples, FACS-sorted cells), the Genomics Platform is now equipped with a SpectraMax Gemini XPS fluorescence-lumi- nescence reader from Molecular Devices
(see system description on Molecular Devices website).
This instrument is used to quantify with high precision and sensitivity nucleic acids in solutions (RNA and DNA) (without prior purification of the samples).
Using fluorescent dyes (Ribo Green™ (RNA) and Pico Green™ (DNA); see a data sheet - PDF; 2 pages, 90,5 KB),
we can detect RNA at 10 ng/ml and DNA at 25 ng/ml.
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