Key Technologies

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ANGIOPREDICT will use a number of key technologies to achieve the project objectives:

 

SNP (Single-Nucleotide Polymorphism) Analysis - SNP analysis of DNA samples will be carried out by VIB using Illumina® SNP Array technology. This array technology allows researchers to analyse samples for hundreds of thousands to millions of markers with dense genome-wide coverage.

Exome Sequencing Analysis - Exome-enriched genomic tumour DNA will be used by UCD to generate raw sequence data using the Illumina® Genome Analyzer II. This approach will provide over 30-fold coverage for at least 90% of all exons.

High-throughput Gene Sequencing - High-throughput gene sequencing data will be generated by UCD using an Agilent SureSelect All Exon kit via use of an exon capturing approach targeted towards the exome.

Mutational analysis - Sequenom® MassArray analysis of genomic tumour DNA will be performed by UCD to validate the cancer selectivity of DNA mutations identified by the high-throughput gene sequencing approach.

Genome-wide DNA Methylation Profiling - Genome-wide DNA methylation profiling will be performed using Differential Methylation Hybridization (DMH) array technology. DNA will be hybridized to a proprietary microarray from EPI that covers 50,000 CpG rich human genomic regions.

RNA Analysis - RNA from patient samples (responders and non-responders) will be isolated, amplified, labeled and hybridized to Affymetrix® Human Gene arrays to correlate gene expression with patient outcome.

FACS (Fluorescence-Activated Cell Sorting) Analysis - Using FACS, single cell suspensions will be generated from patient samples where the cells isolated will be carry specific surface markers. DNA and RNA analysis will subsequently be performed on the isolated cells.

qRT-PCR (Quantitative Real Time Polymerase Chain Reaction) Analysis - qRT-PCR will be used by ONCO to develop a diagnostic assay using predictive gene signatures identified during the project.

H&E (Hematoxylin and Eosin) staining - H&E staining is a staining method commonly used in histology. It is the most widely used approach in medical diagnosis of cancer and will be used for this purpose in the project.

Data Base Integration - SQL (structured Query Language) data bases allow us to store and retrieve above patient-related, genomics, epigenomics and expression data form one source and exploit this information for multivariate statistics and systems biology approaches. Relational data model will be used to structure and interrelate the data ('ontology).

Multivariate statistics - Multivariate statistics methods such as Principal Component or Cluster Analyses allow us to study the effect of several emergent biomarkers in combination. 

Systems Biology approaches: Systems Biology approaches use computational models to study genomic regulation and proteomic pathways on different biological scales. This includes the study of protein signalling in a cell, or the proliferation of tumour and non-tumour cells within a tissue.

DNA copy number alterations - DNA from patient samples (responders and non-responders) will be isolated, and subjected to genome-wide shallow sequence using the Illumina® Genome Analyzer IIx (coverage 0.2x genome). 

RNA Expression analysis from isolated tumor cell compartments - RNA from isolated tumor cell compartments (TEC, TC, TAM) will be isolated, amplified, labeled and hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarray.