Biochip array technology

Cytokines measurements were performed by using the Biochip Array Technology on the Randox Evidence Investigator (Randox Laboratories, Belfast, Northern Ireland). The Evidence Investigator Biochip Array Technology is used to perform simultaneous quantitative detection of multiple analytes from a single patient sample. The applied cytokine array biochip employs a sandwich chemiluminescent immunoassay for a high throughput measurement of circulating cytokines. The light signal generated from each of the test regions on the biochip is detected using digital imaging technology and compared to that from a stored calibration curve. The concentration of cytokines present in the sample is calculated from the calibration curve. The Evidence Investigator Cytokine Array can simultaneously determine the concentrations of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, vascular endothelial growth factor (VEGF), interferon γ (IFNγ), epidermal growth factor (EGF), MCP-1, and TNFα.

Genotyping of NPHSII samples was performed on DNA extracted from blood and carried out using Taqman (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) and KASPar (LGC, Teddington, UK) genotyping assays as well as restriction fragment length polymorphism (RFLP) analysis. For the CoRDia SMI + RR arm, saliva was collected using the Oragene-DNA OG-250 and DNA manually purified using DNA Genotek’s PrepIt-L2P DNA extraction kit (DNA Genotek Inc., Ontario, Canada). Individuals in the CoRDia SMI + RR arm and selected NPHSII samples (n = 185) were genotyped using the Cardiac Risk Prediction Array (Randox Laboratories Ltd, Crumlin, Co Antrim, UK) according to the manufacturer’s instructions. The Cardiac Risk Prediction Array is a multiplex SNP genotyping system which uses Randox’s Biochip Array Technology [13 (link)] to genotype 19 CHD risk SNPs. The protocol involves using multiplex PCR to amplify target DNA in an allele-specific manner. Amplicons are detected by hybridisation to spatially tethered probes on the biochip array surface. Each position on the biochip array corresponds to a specific allele and genotypes are determined using the Evidence Investigator Analyser. In some instances (e.g. for rare genotypes) Sanger sequencing was used to confirm the result.