Applied biosystems 7500 real time pcr instrument

Total nucleic acid was extracted from the whole blood and meat exudate using the MagMAX™ Pathogen RNA/DNA Kit (Life Technologies, Burlington, ON, Canada) and the MagMAX Express-96 Magnetic Particle Processor (Life Technologies) following the manufacturer’s protocol. For the organ samples, nucleic acid extraction was performed on supernatant collected from 10% w/v suspensions in sterile PBS. The ASFV genomic material in the whole blood, organs, and meat exudate was quantified using a previously published TaqMan qPCR assay that specifically amplifies a conserved region of the p72 gene of the virus [45 (link)]. The ASFV qPCR was carried out using the TaqMan™ Fast Virus 1-Step master mix (Life Technologies) on the Applied Biosystems 7500 Real-Time PCR Instrument (Life Technologies) using the cycling conditions recommended for the master mix (50 °C for 10 min, 95 °C for 3 min followed by 40 cycles of 96 °C for 3 sec and 60 °C for 30 sec). The TaqMan RT-qPCR assay for beta-actin developed in-house was used to ensure valid nucleic acid extraction and the absence of PCR inhibitors in the samples [46 (link)]. Samples with Ct values of 35.99 and lower were considered positive, and values between 36 and 40 were considered suspicious.

1 μg of digested RNA was reverse transcribed into complementary DNA with random primers using a GoScript™ Reverse Transcription System (Promega Corporation, Madison, USA), according to the manufacturer’s protocol. Then, qRT-PCR procedure was performed using a GoTaq^® 2-Step RT-qPCR System (Promega Corporation) on a Applied Biosystems 7500 Real-Time PCR instrument (Thermo Fisher Scientific). The reaction was designed as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 59°C for 45 s. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. The sequences of the primers are shown in Table A1. The relative expression level of each gene was calculated using the 2^−ΔΔCT method.

Total RNA was extracted from cultured cells and pulverized frozen tissue by the TRIzol method. For microRNA analysis, all primers were purchased from Quanta BioSciences (Gaithersburg, MD, USA). cDNA was generated using QScript MicroRNA cDNA Synthesis kit (Quanta BioSciences) and q-RT-PCR performed using PerfeCTa SYBR green super mix on a Applied Biosystems 7500 Real-Time PCR Instrument (ThermoFisher), and normalized to RNU6 expression. For mRNA analysis, following column purification using Qiagen RNeasy kit and DNase treatment (Qiagen, Toronto, ON, Canada), cDNA was generated with QScript cDNA super mix (Quanta BioSciences) and analyzed as described above, and normalized to β-actin expression. Primers used were PGC-1α total: Forward 5′-CAGCTTTCTGGGTGGATTGA-3′ and Reverse 5′-GCTCATTGTTGTACTGGTTGGA-3′, Mitofusin-2: Forward 5′-CTCTCAAGCACTTTGTCACTGC-3′ and Reverse 5′-TGTATTCCTGTGGGTGTCTTCA-3′, β-actin: Forward 5′-TTGCTGACAGGATGCAGAAG-3′ and Reverse 5′-TAGAGCCACCAATCCACACA-3′.

According to manufacturer recommended protocols, reference profiles and standard bulk mixture samples were extracted using the QIAamp DNA Ivestigator Kit (QIAGEN, Germantown, MD, USA) and quantified using Quantifiler^® Duo DNA Quantification kit (ThermoFisher Scientific, Carlsbad, CA, USA) on the Applied Biosystems’ 7500 real-time PCR instrument (ThermoFisher Scientific, Carlsbad, CA, USA). One nanogram of DNA extracts were amplified using the GlobalFiler^TM amplification kit (ThermoFisher Scientific, Carlsbad, CA, USA) at 29 PCR cycles. Probabilistic genotyping of donor references profiles and complex equimolar 2-6 person mixtures was conducted using STRmix™ v2.8 and EuroForMix v3.1.0 (Quantitative LR MLE based) according to the known NOC.

DNA extraction was conducted on reference buccal swabs and 60 μL of each mixture cell suspension using the AutoMate Express™ Forensic DNA Extraction System (ThermoFisher Scientific, Carlsbad, CA, USA). Each extraction set contained an extraction blank and was quantified with the Quantifiler^® Duo DNA Quantification kit (ThermoFisher Scientific, Carlsbad, CA, USA) using the Applied Biosystems’ 7500 real-time PCR instrument (ThermoFisher Scientific, Carlsbad, CA, USA).