Whether these regions contain CNVs defined by the Database for Genomic Variation (DGV, http://dgv.tcag.ca/dgv/app/home) , to which both HapMap Project and 1000 Genomes Project are linked (Additional file 1: Figure S1). This screening retrieved 4 locations with CNV loci (Table 1). Two of the locations contained LY2510924 cost overlapping CNVs: esv27061 and esv2757747, and dgv976e1, esv2656635, nsv908562 and dgv986e1 (Table 1).Droplet digital polymerase chain reaction (ddPCR)MethodsParticipantsThe participants included in this study were selected from the Victorian Family Heart Study (VFHS), a healthy population-based cohort of European-descendants collected in Melbourne, Australia, to specifically study the family patterns in cardiovascular risk factors [18-21]. SBP was estimated based in the average of 2 lying SBP values and 2 standing SBP values. SBP and DBP for subjects on antihypertensive treatment (53.1 of the high BP group) were adjusted. Briefly, 10 mm Hg was added to systolic blood pressure and 5 mm Hg to diastolic blood pressure, as previously described [22,23]. In order to maximize the statistical power and enrich for rare variants in the current analysis, we selected a sex-matched sample of biologically unrelated subjects. We selected 96 and 92 subjects from the highest and lowest deciles for SBP in the VFHS, respectively. This was done in consideration of a mean SBP (122 mm Hg; SD: 14.3 mm Hg) in the VFHS, the mean SBPs of the high (166 mm Hg; SD: 12.3 mm Hg) and low (98 mm Hg; SD: 5.2 mm Hg) groups differed by 4.5 SDs. This provides power equivalent to 1714 subjects selected at random . DNA was extracted from white blood cells. All subjects gave informed consent and the study was approved by the human research ethics committee at the Alfred Hospital, Melbourne, Australia. In order to investigate whether having differential number of copies would lead to differential PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27107493 gene expression, we also genotyped forty Polish individuals of white European ancestry from the Silesian Renal Tissue Bank (SRTB), collected to study genetic aspects of human cardiovascular disease. DNA samples were extracted from white blood cells. Recruitment and phenotyping were as described [24,25]. Diagnosis of hypertension was as stipulated for the Silesian Hypertension Study. These subjects underwent elective unilateral nephrectomy because of non-invasive renal cancer. Samples from a pole of kidney unaffected by the neoplastic process had RNA extracted for gene expression analyses. All subjects gave informed consent and theThis study followed the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines . Four CNV assays were selected to genotype the 8 CNVs (Table 1). DNA samples, previously extracted from whole blood [18-21], were quantified by spectrophotometry. Given that one haploid human genome weighs 3.3 pg, and approximately 10,000 copies of genome per reaction are required for accurate ddPCR, we aimed to use 33 ng of DNA per reaction. Prior to PCR, DNA was restriction digested with either XbaI, EcoRI or EcoRV (Promega, Madison, Wisconsin, USA, 1 unit per 1 g of DNA) for 1 hour at 37 , followed by 20 minutes of heat inactivation at 65 in a PCR thermal cycler (BioRad, Hercules, California, USA). PCR reactions were run in a total of 20 l, containing 1 l of the TaqMan assay (Life Technologies, Pleasanton, California, USA) specific for the CNV of interest, labelled with FAM dye, 1 l of the copy number reference assay for the.