Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol, and 5 µg of total RNA was used for complementary DNA (cDNA) synthesis using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA). For quality control, RNA purity and integrity were evaluated using a DS-11 FX UV-Vis spectrophotometer (DeNovix Inc., Wilmington, DE, USA). RNA was then stored at −80 °C prior to real-time quantitative PCR (RT-qPCR) analysis. For quantitative real-time PCR (qPCR), cDNA was mixed with Luna® Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) and specific primers. The qRT-PCR reaction was processed using a magnetic induction cycler (MIC) PCR machine (Bio Molecular Systems, Australia). The relative expression level of a target gene was quantified by normalization with the internal control GAPDH gene, and expression difference was calculated according to the 2−ΔΔCT method. The qRT-PCR primer sequences used in this study are listed in Table 1 .
Mic pcr machine
Manufactured by Bio Molecular Systems
Sourced in Australia
The Mic PCR machine is a thermal cycler designed for the amplification of DNA segments using the Polymerase Chain Reaction (PCR) technique. The device precisely controls the temperature, duration, and cycling of the PCR reaction to facilitate the exponential replication of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Luna universal qpcr master mix, by New England Biolabs (1 mentions)
Dneasy powerlyzer powersoil kit, by Qiagen (1 mentions)
Miracle autoxt automated nucleic acid extraction system, by iNtRON Biotechnology (1 mentions)
4 protocols using mic pcr machine
1
RNA Extraction and qRT-PCR Analysis
2
SARS-CoV-2 RNA Extraction and Quantification
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Viral RNA from nasal swabs was extracted using the Miracle-AutoXT Automated Nucleic Acid Extraction System (iNtRON Biotechnology Inc., South Korea), and viral RNA from fecal samples was extracted using DNeasy PowerLyzer PowerSoil Kit (Qiagen Ltd., GmbH, Germany) following the manufacturer’s instruction. Genes from the PrimerDesign RT-PCR COVID-19 detection kit (PrimerDesign Ltd.) were used for the quantification of the viral RNA in nasal swabs and fecal samples. The primers provided in the kit target the RdRp gene. Internal extraction control primers were also provided to detect the exogenous source of the RNA template added during the extraction step. The PCRs were performed according to the manufacturer protocol using the Magnetic Induction Cycler (MiC) PCR Machine (Bio Molecular Systems, QLD, Australia).
3
Detecting Avian Influenza in Field Samples
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Nucleic acid extracts were tested for AIV by RT-qPCR which amplifies the matrix segment of the virus. Briefly, samples were processed following the AIV Type A generic assay38 (link) with 1 µL of the primer/probe mix (containing 0.9 µM of both forward and reverse primers and 0.25 µM of probe from Heine et al.38 (link)), 5 µL of extracted RNA, 12.5 µL of X2 AgPath-ID One Step PCR RT buffer, 1 µL of X25 AgPath-ID RT PCR enzyme (Thermo Fisher Scientific, MA, USA) and molecular grade water to make up a 25 µL volume. The samples were then run with the following conditions: 45 °C for 60 min; 95 °C for 10 min; and 45 cycles of 95 °C for 15 s, 60 °C for 45 s. For the in-field deployable laboratory, the RT-qPCR assay was conducted on a Mic PCR machine (BioMolecular Systems, New South Wales, Australia) supported by a 12-V battery (Itech 120AH LifePo4) linked to an Itech 2000-W Sinewave inverter and powered by a 100 series 4 × 4 Toyota Landcruiser. AIV positive samples were taken forward for sequencing.
4
Quantitative Analysis of Phytochemical Biosynthesis
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To evaluate the possible effects of rol genes on the phytochemical biosynthesis in MEP, MVA, and shikimate pathways, quantitative real-time PCR (qPCR) of 5 selected genes was performed. PCR conditions were optimized for genes encoding HMGR, HDS, FDS, PAL, and TTG1, accordingly. The amplification reaction was accomplished by gene-specific primers, as shown in Supplementary Table 1. The qPCR was conducted using a Mic PCR machine (Bio Molecular Systems) with 1X Eva Green PCR master mix. For real-time qPCR, a 1:10 dilution of cDNA was used. The reaction conditions for qPCR were as follows: an initial cycle of denaturation for 12 min at 95 °C, followed by 40 cycles each of denaturation for 15 s at 95 °C, primer annealing for 20 s at 62 °C (for all genes), and elongation for 20 s at 72 °C. Two biological samples were analyzed, while three technical replicates were used for each biological sample; the melting curve of amplicons confirmed the absence of primer dimers at the end of each run. The relative gene expression levels were normalized with the endogenous reference gene (β-actin).
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