Openarray accufill 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.

Candidate genes and polymorphisms were identified after browsing the Single Nucleotide Polymorphism Database (dbSNP) and examining the published literature regarding each known gene and variant (favourable and unfavourable) associations^{30 (link)–32 (link)}, and analysing their potential regulatory and biological functions with the Regulome DB and HaploReg v4.1 databases^{33 (link),34 (link)}. On the same day of the anthropometric evaluation, saliva samples were obtained by gently rubbing the inside part of the cheek with a sterile swab, free of human RNA and DNA (Deltalab, Barcelona, Spain). Children were asked to clean their mouths and avoid eating or drinking 60 min before collection of samples to prevent contaminations. Two samples were obtained per student. The swabs were immediately stored in refrigeration and further frozen at −80 °C until their processing. Genomic DNA extraction and genotyping were carried out at the GENYAL Platform (IMDEA-Food, Madrid, Spain) using the OpenArray™ AccuFill™ System (Life Technologies Inc. Carlsbad, CA, USA) as described elsewhere³⁵ . Data analysis was made by TaqMan Genotyper Software v1.3 (autocaller confidence level >90%).

DNA was obtained from saliva samples collected the same day of the anthropometric evaluation. Genomic DNA was extracted according to the protocol described by Stratec^® INVISORB^® Spin Tissue Mini Kit (INVITEK Molecular GMBH, Berlin, Germany). For genotyping, the DNA samples were loaded in TaqMan^® OpenArray^® Real-Time PCR plates (Life Technologies Inc., Carlsbad, CA, USA) already configured with the specific selected SNPs with specific waves for each allele marked with a different fluorophore to determine the genotype. This process was made using the OpenArray^® AccuFill™ System (Life Technologies Inc., Carlsbad, CA, USA). Once it was ready to be used, a PCR was run, and the chips were read in the QuantStudio^® 12K Flex Real-Time PCR instrument (Life Technologies Inc., Carlsbad, CA, USA). The results were analyzed using the TaqMan^® Genotyper software (Life Technologies Inc., Carlsbad, CA, USA), which assigns the genotype automatically to each sample according to the amount of detected signal for each fluorophore. Data analysis was made by TaqMan Genotyper Software v1.3 (autocaller confidence level > 90%). Call rates for all SNPs were >96%, and genotype frequencies were in Hardy-Weingberg equilibrium (p > 0.05).