Genotyping and expression

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.

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.

The new QuantStudio 7 Pro system is a 7th generation real-time PCR system that enables high qPCR productivity thanks to its flexibility, specificities and performance.

1°) Determination of the concentration of a nucleic target thanks to a control concentration curve (standard curve). Use of specific TaqMan probes or SYBR Green.

2°) Determination of the relative concentration of a sample or a target thanks to a standard curve and an endogenous control. Concentration measurements are thus normalized using the endogenous control. Use of specific TaqMan probes. This method can be used to :

• compare the expression of a gene in different tissues.
• compare the expression of a gene in a treated and an untreated sample.
• compare the expression of a gene with the wild-type allele versus the mutated allele.
• compare the expression of a gene as a function of time under different treatment conditions.

3°) Determination of the genotype of a genetic variant (SNP or Insertion/Deletion) either in real time (Real Time PCR) or at the end of the PCR reaction. Use of specific TaqMan probes.

4°) Determination of the presence or absence of the expression of a gene in a sample. Realization of a PCR with two TaqMan probes specific to an internal control and to the gene of interest.

5°) Determination of the melting temperature of a nucleic target (TM). Realization of a melting curve by a temperature gradient in the presence of SYBR Green at the end of the PCR. Determination of the presence or absence of contaminating amplificates.

For each point, the study can be done either on 96 samples or on 384 samples.

The PamStation 12 is a fully automated instrument designed for processing PamChip 4 microarrays. It is available with either phosphotyrosine kinase (PTK) or serine-threonin kinase (STK) arrays. The 3D surface of the PamChip microarrays provides unique characteristics to allow real-time analysis of nucleic acid and protein binding for allows enzymatic reactions of kinases to be followed kinetically.

The sample consisting of protein and labelled antibodies are dispensed onto the PamChip, which is placed in the PamStation12. Once inside the instrument the sample is hybridized or incubated at a pre-determined temperature. During hybridization or incubation the sample is pumped back and forth through the porous material to maximize binding kinetics and minimize assay time. Reaction times are typically minutes to an hour.