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T1000 thermal cycler

Manufactured by Bio-Rad
Sourced in United States, Australia

The T1000 thermal cycler is a compact and efficient instrument designed for performing polymerase chain reaction (PCR) experiments. Its core function is to precisely control the temperature of samples during the various stages of the PCR process, enabling the amplification of target DNA sequences.

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12 protocols using t1000 thermal cycler

1

Genotyping of brclf Mutant

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An EMS-induced TILLING mutant brclf (line number: ji32391-a) was purchased from RevGenUK (Stephenson et al., 2010). Genomic DNA from individual brclf mutants were extracted using DNeasy Plant mini kit (QIAGEN, Germany) and used for PCR amplification. Gene-specific primer pair (BrCLF_genoF and BrCLF_genoR) were used for genotyping of brclf mutant using T1000™ thermal cycler (Bio-rad, USA). Amplified PCR fragments (about 500bp) were run on 1% agarose gel and extracted from gel by using a QIAquick Gel extraction kit (QIAGEN, Germany) and then confirmed by the direct Sanger sequencing (Bionics Co., South Korea). Information on primer sequences is presented in Supplementary Table S1.
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2

Total RNA Extraction and cDNA Synthesis

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Total RNA was extracted using 1 mL TRI reagent (Sigma-Aldrich, Castle Hill, NSW, Australia) and 0.2 mL chloroform and precipitated with 0.5 mL 2-propanol (Sigma-Aldrich). Pellets were washed twice with 75% ethanol and air-dried. RNA concentrations were calculated using NanoDrop™ 2000 (ThermoFisher Scientific). A total of 1 µg of total RNA were loaded in each cDNA synthesis reaction. cDNA synthesis was conducted using the T1000 thermal cycler (Bio-Rad, Gladesville, NSW, Australia) in a final volume of 20 µL. Each reaction contained 1 μg of RNA diluted in a volume of 11 µL, to which we added 9 µL of cDNA synthesis mix (Tetro cDNA synthesis kit) (Bioline, Australia). Samples were incubated at 45 °C for 40 min followed by 85 °C for 5 min. Finally, cDNA samples were stored at −20 °C until use.
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3

Mycelia RNA Extraction and cDNA Synthesis

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Approximately 50 mg of mycelia was grounded with poly(vinylpolypirrolidone; 0.4 mg per mg of mycelia) with a mortar and pestle. The final powder was used in the extraction and purification of total RNA using the RNeasy Plant Mini Kit (QIAGEN), according to the manufacturer’s protocol. Genomic DNA digestion was done with the RNase‐Free DNase Set (QIAGEN). Quality, integrity and quantity of the total RNA were analysed in a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by running 2 μg of RNA into 1% agarose gels in TBE (Tris‐boric acid‐EDTA) buffer. The complementary DNA (cDNA) was synthesized from 1000 ng of the total RNA using an iScript cDNA Synthesis Kit (Bio‐Rad) in T1000 Thermal Cycler (Bio‐Rad, Hercules, CA, USA). The reaction protocol consisted of 5 min at 25 °C, 30 min at 42 °C and 5 min at 85 °C.
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4

Quantification of TREM-1 and TREM-2 Expression in Tendon Tissue

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Total RNA (1μg) from tendon tissue was reverse transcribed to cDNA using PROMEGA cDNA synthesis kit according to the manufacturer’s protocol. The RNA and oligo-dT primers were primed prior to the reaction. Reaction mixture (MgCl2, reaction buffer, dNTP mix, RNase inhibitor and reverse transcriptase) was prepared for 20μl reaction volume and cDNA synthesis was carried out in Bio-Rad T100 thermal cycler. Gene expression levels of TREM-1 and TREM-2 were quantified by the real-time PCR system (Bio-Rad T1000 thermal cycler). The 8μl cDNA (1:1 dilution) was mixed with 10μl SYBR Green PCR master mix (PROMEGA) and corresponding forward and reverse primers (10pM/μl) (Table 1) for real-time PCR amplification. The 18s rRNA was used as the housekeeping gene. The reaction conditions were 95°C for 10 minutes, 40 cycles at 95°C for15s, and 60°C for 1 min. The mRNA expression of TREM-1 and TREM-2 was normalized with the expression level of 18s rRNA. Four independent experiments were carried out for each sample and average of Cq values was taken. Results are represented as fold-change of TREM-1 and TREM-2 mRNA levels between patients and the reference gene [41 (link)].
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5

Nested PCR for Piroplasmid 18S rDNA

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A nested PCR (nPCR) targeting a 561 bp fragment of the 18S rDNA nuclear gene of piroplasmids was employed. The primers used for the nPCR were the following: BTH-1 F (5′-CCT GAG AAA CGG CTA CCA CAT CT-3′), and BTH-1R (5′-TTG CGA CCA TAC TCC CCC CA-3′) for the first round and GF2 (5′-GTC TTG TAA TTG GAA TGA TGG-3′), GR2 (5′-CCA AAG ACT TTG ATT TCT CTC-3′) for the second round. For PCR amplification the T1000™ Thermal Cycler (Bio-Rad, London, UK) was used with the following conditions: initial denaturation at 95°C for 3 min, then 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s (for the first round), 50°C for 30 s (for the second round) and extension at 72°C for 1 min (for the first round), 72°C for 40 s (for the second round) and a final extension at 72°C for 7 min (18 (link)). During the protocol, two negative controls (PCR water) were used for checking the possible contamination and one positive control consisting in DNA isolated from the blood of a naturally infected dog with Babesia canis (confirmed by sequencing) were included in each amplification set.
Amplification products were visualized by electrophoresis on 1.5% agarose gel stained with RedSafe™ 20,000 × Nucleic Acid Staining Solution (Chembio, UK), and their molecular weight was assessed by comparison to a molecular marker (HyperLadderTM 100 bp, Bioline, London, United Kingdom).
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6

Quantification of Circulating Biomarkers

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ddPCR assays performed on cfDNA and EV‐RNA were from Bio‐Rad Laboratories Inc (ERBB2: dHsaCP1000116; dHsaCPE5037554, EIF2C1: dHsaCP2500349, EEF2: dHsaCPE5050049) and PCR reactions were run on T1000 thermal cycler (Bio‐Rad, Hercules, CA, USA). Analysis of ddPCR data was analyzed using QuantaSoft Software, version 1.7 (Bio‐Rad Laboratories, Inc.). EV‐RNA isolation was performed as detailed in Supplementary Materials and Methods.
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7

Quantitative Gene Expression Analysis

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Total RNA was isolated and extracted using TRIzol reagent (ThermoFisher Scientific, 15596026). RNA content was quantified using a NanoDrop200 (Thermo Scientific). RNA (1 μg) was reverse transcribed iScript gDNA Clear cDNA kit (Bio-Rad, 1725035) and T1000 Thermal Cycler (Bio-Rad). Gene expression was measured using 10 ng cDNA, SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725271) and CFX Connect Real Time PCR System (Bio-Rad) as per manufacturer’s protocols. Primer sequences were obtained from the Harvard Primer Bank and purchased from Integrated DNA Technologies (IDT). Forward and reverse sequences of genes utilized in this study can be found in Table 1.
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8

Digital PCR for HER2 Amplification

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Digital PCR was performed using the droplet digital PCR (ddPCR) method on Bio-Rad QX200™ (Bio-Rad, Hercules, CA, USA). A total 20 µl PCR reaction was prepared with 15–20 ng DNA and 2X ddPCR Supermix for probe (Bio-Rad, Hercules, CA, USA); primers and fluorescent probes (FAM and VIC) were prepared from Prime PCR assay for ddPCR (dHsaCP1000116 for HER2 and dHsaCP2500349 for EIF2C1 as the reference control). HindIII was mixed in PCR reaction. Droplets were generated by Bio-Rad QX200 droplet generator. Then, the total 40 µl of emulsified PCR reactions were transferred to a 96-well plate and heat sealed before running on T1000 thermal cycler (Bio-Rad, Hercules, CA, USA) with the following cycle: 95 °C for 10 min, 40 cycles of 94 °C for 30 s and 60 °C for 60 s, 98 °C for 10 min, and hold at 4 °C. The temperature ramp rate was 2°C/s for all steps. Negative control with no DNA was included in each run. After the PCR, the PCR plates were transferred to Bio-Rad QX200 droplet reader. Analysis of ddPCR data was performed by using QuantaSoft v1.3.2.0 software from Bio-Rad. HER2/EIF2C1 ratio ≥ 2.0 was defined as HER2 amplification, and HER2/EIF2C1 ratio < 2.0 was defined as HER2 non-amplification.
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9

ITS2 Amplification and Illumina Sequencing

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Library preparation was performed by amplification of ITS2 region using primer set described by White et al.61 with Illumina's adapters attached (ITS3: 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GCA TCG ATG AAG AAC GCA GC-3′; ITS4R: 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GTC CTC CGC TTA TTG ATA TGC-3′). PCR reactions were conducted with the use of Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and 100 ng of total DNA in T-1000 Thermal Cycler (BioRad, Hercules, California, United States). Reaction conditions were as follows: initial denaturation at 98 °C for 30 s, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 20 s and elongation at 72 °C for 15 s, and at the end 10 min elongation step at 72 °C. Illumina MiSeq in 2 × 300 bp paired-end format was performed for ITS2 sequencing with the use of MiSeq Reagent Kit v3 (Illumina, California, United States). Second stage of library preparation, sequencing of 120 samples and sequence pre-processing was conducted in Genomed company (Warsaw, Poland).
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10

Total RNA Extraction and cDNA Synthesis

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Total RNA was extracted from untreated BV2 cells (Ctrl) or cells exposed to 1000 µM AZE for 6 and 24 h, respectively. Briefly, cells were lysed using 1 mL TRI reagent (Sigma-Aldrich, Castle Hill, NSW, Australia) and 0.2 mL chloroform and precipitated with 0.5 mL 2-propanol (Sigma-Aldrich) [20 (link)]. Pellets were washed twice with 75% ethanol and air-dried. RNA concentrations were calculated using NanoDrop™ 2000 (ThermoFisher Scientific, Waltham, MA, USA). A total of 1 µg of total RNA were loaded in each cDNA synthesis reaction. cDNA synthesis was conducted using the T1000 thermal cycler (Bio-Rad, Gladesville, NSW, Australia) in a final volume of 20 µL. Each reaction contained 1 μg of RNA diluted in a volume of 11 µL, to which we added 9 µL of cDNA synthesis mix (Tetro cDNA synthesis kit; Bioline, Redfern, NSW, Australia). Samples were incubated at 45 °C for 40 min followed by 85 °C for 5 min. Finally, cDNA samples were diluted at a final concentration of 10 ng/mL in milliQ H2O and stored at −20 °C until use.
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