Genotyping and expression

Our laboratory is equipped with technologies dedicated to the most efficient and innovative genomics. We are thus able to answer to all requests in this area.

iScan – Illumina

The iScan array scanner enables a wide variety of applications for superior genetic analysis studies, including:

  • Genotyping : Illumina genotyping microarrays allow for powerful genome-wide association studies.
    Genotyping applications include:
        1. SNP genotyping and CNV analysis
        2. Linkage analysis
        3. Targeted genotyping
  • Gene Expression Analysis : Gene expression arrays provide a global view of gene activity within cells. BeadArray technology enables robust and accurate measurement with wide dynamic range for large gene expression studies.
  • FFPE Sample Analysis : Formalin-fixed, paraffin-embedded (FFPE) samples are preserved tissue samples that generally contain partially degraded RNA, making transcription analysis more challenging. Illumina offers solutions to provide high-quality array data from degraded RNA samples.
  • Methylation Analysis : Epigenetic arrays let you quantitatively scan for methylation sites across the genome, utilizing high-multiplex assays for maximum throughput. Our Infinium Methylation Assay covers CpG islands, miRNA promoter regions, and other areas important in epigenetic regulation.

Odyssey – LICOR

Odyssey is an infrared imaging system which enables precise quantification over a range of about 4 log, and an increase of the sensitivity due to the absence of background noise. Odyssey is equipped with two lasers, allowing detection simultaneously on two wavelengths (around 700 and 800 nm). Immediate applications are standardizing results on the same matrix with the housekeeping gene, or the simultaneous detection of a protein and its phosphorylated form.

Finally, in addition to the “classic” western, the Odyssey can be used for many other applications like “in-cell” immunodetection experiments to quantify a protein on fixed cells in 24 or 96 micro-wells. This allows the study of the signaling pathways and screening without artifact due to cell lysis.

We can also count directly cells with a nuclear marker like DRAQ5.

Glomax multi – Promega

The GloMax®-Multi Detection System can function as a luminometer (luciferase activity measurements) or a photometer (protein concentration quantification) in a 96 well plate format. Fast measurements, easy to use. Files can be exported in a .csv format.

QX200 Droplet digital PCR system – BIORAD

Droplet Digital PCR (ddPCR™) System provides absolute quantification of target DNA or RNA molecules for EvaGreen or probe-based digital PCR applications. The emerging applications are genomic alterations as gene copy number variation, NGS libraries quantification, absolute quantification, detection of rare sequences and rare mutations, gene expression and microRNA analysis, single cell analysis. The QX200 droplet generator partitions samples into 20,000 droplets. After PCR, droplets are read on a QX200 droplet reader in two hours, which counts the fluorescent positive and negative droplets to calculate target DNA concentration.

BioMark HD – Fluidigm

BioMark HD – Fluidigm : The integrated fluidic circuits (IFCs) allow automating PCR reactions in nanoliter volumes, so you use less sample and reagent. The Biomark HD system runs in either real-time or end-point read modes, bringing flexible, efficient and economical PCR solutions to a range of applications.In the lab we use 4 IFC :

  • 96.96 : 96 targets against 96 samples at once.
  • 48.48 : 48 targets against 48 samples at once.
  • 192.24 : 24 targets against 192 samples at once.
  • FLEXsix : six partitions (12 assays by 12-samples) that can each be run separately or together.

With these 4 IFC, we can do Gene expression and Genotyping studies. We also study at the lab the polymorphism : Copy number variation (CNV). The Digital Array IFC works by partitioning a single sample into 765 or 770 individual 6 nL real-time qPCR reactions. The concentration of any sequence in a DNA sample (copies/μL) can be calculated using the number of positive chambers that contain at least one copy of that sequence. For this type of study we have 2 types of IFC :

  • 765 : 12 samples into 765 chambers
  • 770 : 48 samples into 770 chambers

nCounter analysis – Nanostring

nCounter analysis – Nanostring : The nCounter® Analysis System from NanoString Technologies offers a simple, cost-effective way to profile hundreds of mRNAs, microRNAs, or DNA targets simultaneously with high sensitivity and precision. Digital detection of target molecules and high levels of multiplexing eliminate the compromise between data quality and data quantity, producing gold-standard sensitivity and reproducibility for studies of hundreds of targets. NanoString’s system uses molecular “barcodes” to detect and count hundreds of unique transcripts in a single reaction. Unlike other methods, the protocol does not include any amplification steps that might introduce bias to the results. The systeme is divided in two parts:

  • The nCounter Prep Station: It processes nucleic acid samples post-hybridization to prepare them for data collection on the nCounter Digital Analyzer. Prior to placing samples on the Prep Station, samples are hybridized according to the nCounter protocol. On the deck of the Prep Station, hybridized samples are purified and subsequently immobilized in the sample cartridge for data collection. The Prep Station can process up to 48 samples in a 10-hour time span.
  • The nCounter Digital Analyzer: it collects nucleic acid data by taking images of the immobilized fluorescent reporters in the sample cartridge with a CCD camera through a microscope objective lens. Images are processed internally and the data output files include the target identifier and count number along with a comprehensive tally of internal controls that allows each assay to be quantitative.

Seahorse XFe24 – Seahorse Bioscience

Seahorse XFe24 analyzers simultaneously measure mitochondrial respiration (oxygen consumption) and glycolysis (the release of H + ion) using non-invasive optical microsensors. Featuring a transitional microchamber the XFe24 allows for accurate measurements, non-invasive, non-destructive on 24 samples simultaneously with the sequential automatic injection of 4 drugs to specifically study different mitochondrial complexes, for example. One advantage is that the XFe24 is usable not only on monolayers of adherent primary cells (differentiated or not), cell lines, non-adherent cells, on isolated mitochondria but also on isolated pancreatic islets allowing accurate analysis of mitochondrial function in different tissue environments.

Viia7 Real Time PCR System – Life Technologies

Viia7 Real Time PCR System – Life Technologies : The new ViiA™ 7 System is a 7th generation real-time PCR system that enables high-productivity qPCR through flexibility, and performance.

  • 1°) Calculation of the absolute target quantity in samples with a standard curve. This method is able to determine the concentration of a target in samples and in a standard dilution series. Spécific TaqMan Gene Expression Assays ou SYBR Green reagents can be used.
  • 2°) Determination of the relative concentration of a sample or a target thanks to a standard curve and an endogenous control. The measures of concentrations are so normalized by using the endogenous control. Relative Standard Curve experiments are commonly used with spécific TaqMan Gene Expression Assays to:
    • Compare expression levels of a gene in different tissues.
    • Compare expression levels of a gene in a treated sample and an untreated sample.
    • Compare expression levels of wild-type alleles and mutated alleles.
    • Analyze the gene expression changes over time under specific treatment conditions.
  • 3°) Genotyping Experiments. Study of genetic variations (Single Nucleotid Polymorphism or Insertion/ Deletion) with spécific TaqMan Gene Expression Assays.
  • 4°) Determination of the presence or the absence of the expression of a gene in a sample. Performed PCR with two probes TaqMan specific to an internal control and to a gene of interest.
  • 5°) Determination of the melting temperature of a nucleic acid sequence (TM). We performed with SYBR Green reagents a melting curve thanks to a temperature gradient at the end of the PCR. Determination of nonspecific PCR amplification.

Each study can be performed with 96 or 384 samples

TLDA – Life Technologies

TLDA means TaqMan Low Density Array and allows you to measure gene expression using the comparative CT method of relative quantitation. It’s possible to study plenty of genes or miRNA on severals samples in a very small reaction volume (2 µl: 1 µl of sample and 1 µl of reaction mix).

Two types of 384 wells TaqMan Array cards are available:

  • Customized cards containing the lyophilized Taqman assays of interest.
  • Predefined cards for medical or scientific diagnostics.