Taqman openarray system

The expressions of 18 genes (Table 3) were evaluated using real-time PCR. Three genes were used as endogenous controls to calculate the relative expressions of the other 15 candidate genes. The selection of reference and target genes was based on results observed in previous transcriptomics studies, where the administration of rbST and anabolic agents to cattle was evaluated [13 (link),14 (link),15 (link),16 (link),17 (link),31 (link)]. Gene-expression assays were carried out with a TaqMan^® OpenArray^® system (Applied BiosystemsTM, Thermo Fisher Scientific), involving a nanoliter high-throughput real-time PCR platform where 3072 reactions were performed simultaneously in the same OpenArray^® plate, and the primers and TaqMan^® probes were preloaded in the plates by the company. A plate design of 18 assays in triplicate, and 56 samples was chosen. Real-time PCR reactions were performed according to the TaqMan^® OpenArray^® protocol. Briefly, in a 384-well plate, 1.2 µL of each cDNA sample was mixed with 3.8 µL of TaqMan^® OpenArray^® Real-Time PCR Master Mix (Applied BiosystemsTM, Thermo Fisher Scientific). The PCR reaction mixtures were loaded automatically into the OpenArray^® plates using an OpenArray^® AccuFill™ System (Applied BiosystemsTM, Thermo Fisher Scientific). The following real-time PCR protocol was used: 40 cycles at 95 °C for 15 s, and 60 °C for 1 min.

The seven CRP SNPs studied (rs11265257, rs1130864, rs1205, rs1800947, rs2808632, rs3093077 and rs876538) were selected on the basis of previous publications in rheumatic diseases [16 (link),18 (link),25 (link)] (Figure 1). The nucleotides are stated according to the plus strand. Genotyping was analyzed using the TaqMan OpenArray system from Applied Biosystems (Foster City, CA, USA). We typed one SNP with a custom-designed genotyping assay and six SNPs with a predesigned assay from Applied Biosystems (see Additional file 1: Table S1 for assay information). We have previously described this method in more detail [26 (link)]. OpenArray plates were manufactured by Applied Biosystems. A nontemplate control was introduced within each set of assays. TaqMan OpenArray Master Mix was used according to the manufacturer’s protocol. Samples were loaded into OpenArray plates using the OpenArray NT Autoloader and cycled using the GeneAmp PCR System 9700 thermal cycler with PCR conditions set according to the manufacturer’s protocol. The arrays were read using the OpenArray NT Imager, and the allele calls and scatterplots were generated with the genotyping software associated with the OpenArray system.

SNPs were selected considering previously published data suggest their association with pediatric-onset T2D in Mexicans (Table 1). DNA was extracted from peripheral blood leukocytes using commercial kits following the manufacturer’s instructions (QIAmp96 DNA Blood, Mini/Kit, Qiagen, Germany). Purity and concentration were assessed through spectrophotometry at 260/280 nm (Epoch spectrophotometer, BioTek Instruments, Winooski, VT, USA), and the integrity was checked following 0.8% agarose gel electrophoresis. SNPs were genotyped using the TaqMan^® OpenArray^® system (Applied Biosystems, Foster City, CA, USA).