Quantstudio 12k flex real time pcr system

The GenoPharm^® open array panel uses TaqMan Assay chemistry for the polymerase chain reaction (PCR) using the Applied Biosystems^™ QuantStudio^™ 12K Flex Real-Time PCR System (Applied Biosystems, Singapore). DNA samples were diluted to 50 ng/μL using nuclease-free water (Ambion^® Cat No. AM9930) and added to a 384 well plate. A reaction mixture of 5 μl genomic DNA and 5 μl of TaqMan^™ Genotyping master mix (Cat. No. 4462164) was prepared in a 96-well plate. The plate was covered with adhesive PCR Foil (Thermo Fisher) and centrifuged for 1 min at 500g. The mixture was transferred to the GenoPharm^® open array panel using the automated Applied Biosystems^™ QuantStudio^™ 12K Flex OpenArray^™ AccuFill^™ System according to manufacturer’s instructions. A no template control (NTC; reaction mixture with all reagents but no template DNA) was included in each run. The reaction mix of 33nl of DNA per data point was run on the Applied Biosystems^™ QuantStudio^™ 12K Flex Real-Time PCR System. Genotypes for the samples were determined by the TaqMan^™ Genotyper Software as per manufacturer’s instructions. All calls were made at cycle 40 using the default quality value of QV ≥ 0.95 to assign a genotype call. Samples were run in triplicate.

The aim of this study was to examine the impact of polymorphisms in ND-related genes on their mRNA expression levels. For genotyping SNPs, we used qPCR with TaqMan assays and used the StepOnePlus Real-Time PCR System (Life Technology, Carlsbad, CA, USA) with TaqMan OpenArray DNA microchips for the QuantStudioTM 12K Flex Real-Time PCR System. The resulting genotyping data were analyzed using Genotyper software (Thermo Fisher Scientific, Waltham, MA, USA).

SNPs were identified using the International HapMap Project SNP database, based on the National Center for Biotechnology information (NCBI) B36 assembly Data Rel 28, phase II + III, built 126. The Gene Tagger procedure in Haploview v4.2 was used to identify tagging SNPs with a minor allele frequency (MAF) >5% and pairwise tagging (r² ≥ 0.80) located in gene regions and surrounding regions (2 kb upstream and downstream gene). The mean LD (r²) between SNPs was 0.96 for IQCJ, 0.96 for NXPH1, 0.97 for PHF17 and 0.95 for MYB. SNPs were selected in a way to cover ≥85% of all common variations (MAF > 5%). The GenElute Gel Extraction Kit (Sigma-Aldrich Co., St. Louis, MO, USA) was used to extract genomic DNA (gDNA) from blood samples. Tagged SNPs were genotyped using TaqMan technology: 2.5 µL of each gDNA (40 ng/µL) and 2.5 µL of OpenArray Genotyper Master Mix (Life Technologies, Carlsbad, CA, USA) were mixed in a 384-well plate with validated primers and loaded onto genotyping plates using the QuantStudio^TM 12K Flex OpenArray^® AccuFill^TM System (Life Technologies). The QuantStudio^TM 12K Flex Real-Time PCR System (Life Technologies) was used for genotyping. Finally, TaqMan Genotyper v1.3 (Life Technologies) was used to call genotypes and export data.

In a final volume of 20 µL, 5 ng of cDNA were mixed with 10 µL of SYBR Green® mastermix (Applied Biosystems) and 300 nM of forward and reverse primers. The reaction was programmed on a QuantStudio^TM 12 K Flex Real-Time PCR System (Life Technologies) as follows: 50 °C for 2 min, 95 °C 5 min, followed by 40 cycles (95 °C for 15 sec and 60 °C for 1 min). The primer sequences are indicated in Supplementary Table 11. To ensure a reliable quantification of transcripts in bovine skin, the stability of three genes (RPL32, GAPDH and β2-microglobulin) was tested in a set of 11 samples (from two dominant black, two recessive red and seven dominant red Holstein animals). Their expression stability was calculated using the ExpressionSuite software (Life Technologies), and RPL32 was considered as the less variable gene (score 0.474) compared to GAPDH (score 0.497) and β2-microglobulin (score 0.643). The target transcripts were further quantified using RPL32 as a reference gene. The relative quantification of each transcript was calculated with the ΔΔCt method of the ExpressionSuite software (Life Technologies). Transcript expression levels of transcripts in one black Holstein individual were set to a value of 1, and the other expression values were calculated relative to this reference.

Viral DNA was extracted from the EDTA-treated blood sample using a Nucleospin^® Blood L kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer’s instructions. Viral DNA was extracted from 2 × 10⁶ PBMCs, 140 µL of viral inoculum stock, 140 µL of clarified nasopharyngeal swabs and 140 µL of cell culture lysate using QIAamp^® Viral RNA Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. EHV-1 ORF30 C₂₂₅₄ mutation in the viral inoculum stock and absence of EHV-1 strains with G₂₂₅₄ and A₂₂₅₄ mutations, EHV-4, EHV-2, EHV-5 and equine influenza virus was confirmed by qPCR before infection. qPCR for EHV-1, EHV-4, EHV-2, EHV-5 and equine influenza virus was performed as previously described [56 (link)]. Typing EHV-1 qPCR (G₂₂₅₄/A₂₂₅₄/C₂₂₅₄) was performed as described previously [15 (link)]. Each reaction was performed in a total volume of 25 µL containing 12.5 µL of 2× Taqman^® Universal Master Mix (Life Technologies, Villebon-surYvette, France), 1.25 µL of primers, 1.5 µL of probes, 6 µL of nuclease-free water and 2.5 µL of template DNA using QuantStudio^TM 12 K Flex Real-Time PCR System (Life Technologies). The limit of detection (LOD 95%) for EHV-1 qPCR [57 (link)] is 7.2 copies/µL of reaction. Below this threshold, the accuracy of signal quantification is reduced. Sequences of primers, probes and thermal cycling are described in Table S3.

Total RNA was extracted using TRIzol reagent (Invitrogen, cat# 15596026). RNA was quantified spectrophotometrically based on the A260 value in an ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). mRNA was reverse-transcribed to cDNA using a cDNA kit (Takara). cDNA was amplified in a mixture reaction system containing SYBR Green qPCR SuperMix-UDG (Takara) and specific primer sequences (Supplementary Table 1). The qRT-PCRs were run on a QuantStudio^TM 12 K Flex Real-time PCR system (Life Technologies, Thermo Fisher Scientific).

We sought to investigate the effects that polymorphisms and genetic variation in different NDD-related genes have on their mRNA expression levels. SNPs and copy number variants (CNVs) were genotyped by qPCR with Taqman assays using the Step one Plus Real Time PCR System (Life Technology, Waltham, MA, USA) and Taqman OpenArray DNA microchips for the QuantStudioTM 12K Flex Real-Time PCR System. Genotyper software (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the genotyping data.