HPMEC cells were grown and treated with human whole blood as above. RNA was isolated using the QIAGEN RNA mini kit; cDNA synthesis was carried out using the High-capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions. For each sample, RNA was reverse-transcribed using a T-Gradient Thermoblock (Biometra). qPCR was conducted using power SYBR Green PCR master mix. cDNA was denatured at 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds then 6 0°C for 1 minute, qPCR was performed with the ABI prism 7900 HT (Applied Biosystems), and the data were analyzed with SDS software v 2.1 (Applied Biosystems). Relative gene expression was compared using the comparative CT method. The primers for DARC and 18S rRNA are described in the Key result table. A fixed amount of cellular cDNA was added to each reaction so that 18S rRNA could be used as a reference.
T gradient thermoblock
Manufactured by Analytik Jena
Sourced in Germany
The T-Gradient Thermoblock is a laboratory equipment designed for precise temperature control. It features a multi-well block that can maintain a temperature gradient across multiple samples simultaneously, allowing for efficient optimization of experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Rna mini kit, by Qiagen (1 mentions)
Sds software v2, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (1 mentions)
Taq pcr starmix with loading dye, by GenStar (1 mentions)
Gelview, by BioTeke (1 mentions)
Taq pcr master mix kit, by Qiagen (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
100 bp dna ladder, by Takara Bio (1 mentions)
Topscript cdna synthesis kit, by Enzynomics (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Tiangel midi purification kit, by Tiangen Biotech (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Reverse transcriptase, by Takara Bio (1 mentions)
Kapa sybr fast qpcr reagent, by Roche (1 mentions)
Miniopticon real time pcr system, by Bio-Rad (1 mentions)
Improm 2 reverse transcription system, by Promega (1 mentions)
13 protocols using t gradient thermoblock
1
Quantitative RT-PCR Analysis of DARC Expression
2
PCR Amplification and Sequence Analysis
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Polymerase chain reaction (PCR) amplification was conducted in a 20-µL reaction volume mixture containing 10 µL 2× Taq PCR StarMix with Loading Dye (GenStar BioSolutions Co., Ltd., Beijing, China), 10 μm of each oligonucleotide primer, 2 µL template DNA, and 6 µL double distilled water. For duplex PCR, two pairs of primers were added and the amount of distilled water was reduced to 4 µL; other ingredients remained unchanged. The thermal cycling steps consisted of an initial pre-denaturation at 94 °C for 2 min, followed by 35 cycles of denature at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension of 72 °C for 5 min. The PCR amplification was carried out in a T-Gradient Thermoblock (Biometra, Göttingen, Germany). The PCR products (7 µL per reaction) were electrophoresed in 2% Tris-acetate-EDTA–agarose (Biowest, Shanghai, China) gel containing 0.01% Gelview (BioTeke, Beijing, China). The agarose gel was visualized and photographed under UV light using automatic gel imaging analyzer (JS-6800, Shanghai Peiqing Science & Technology Co., Ltd., Shanghai, China). The positive products were recycled and then sequenced from both ends using an ABI 3730XL automatic sequencing system (TSINGKE, Hangzhou, China). Sequence specificity analyses for the MRJP2 fragment were performed using BLAST (http://blast.ncbi.nlm.nih.gov/ ) (National Library of Medicine, Bethesda, MD, USA).
3
Genomic DNA Extraction and Sequencing
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Genomic DNA of all investigated strains was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Nucleotide sequences of the SSU rRNA gene together with ITS‐1‐5.8S‐ITS‐2 region were amplified using a set of Taq PCR Mastermix Kit (Qiagen GmbH) and a complex of EAF3 and ITS055R as well as algal‐specific primers G800R and G500F. PCR reactions were made in a thermocycler T gradient Thermoblock (Biometra, Analytik Jena, Germany) under conditions described in a previous paper (Mikhailyuk et al. 2018 ). PCR products were cleaned using a Qiagen PCR purification kit (Qiagen GmbH) according to the manufacturer's instructions. Cleaned PCR products were sequenced commercially by Qiagen Company using primers G800R, 536R, 920F, 1400R, 1400F, GF, GR, and ITS2F. Sequences of all primers used in the study with respective references are included in the Table 2 . The resulting sequences were assembled and edited using Geneious software (version 8.1.8; Biomatters). They were deposited in GenBank under accession numbers MN267182 – MN267185.
4
Citrus Leaf RNA Extraction and RT-PCR
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Total RNA was extracted from approximately 20 mg of young citrus leaves using TRIzol Reagent (catalog no. 15596-026; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA was dissolved in 100 μl FORMAzol solution (Molecular Research Center, Cincinnati, OH, USA) for stabilization of RNA solubilization and stored in −20°C or −70°C. RT-PCR was performed as a two-step procedure, which included RT, using a TOPscript cDNA synthesis kit (catalog no. EZ105S; Enzynomics, Daejeon, Korea) for synthesis of cDNA according to the manufacturer’s protocol. PCR was carried out with primer sets (Table 2 ) using Accupower Hotstart PCR pemimix (catalog no. K-5051; Bioneer, Daejeon, Korea). Amplification was performed in a programmable thermocycler (model T-Gradient Thermoblock; Biometra, Göttigen, Germany), using an initial denaturation step of 95°C for 2 min, followed by 35 amplification cycles of denaturation at 94°C for 30 s, annealing at 50–55°C for 1 min, and extension at 72°C for 2 min, and a final extension at 72°C for 10 min. After PCR, 12 μl of the product was electrophoresed in a 1.0% agarose gel in 1× TAE buffer for 30 min at 3 V/cm and visualized by ethidium bromide staining. A 100-bp DNA Ladder (catalog no. 3407A; TaKaRa, Tokyo, Japan) was used for molecular weight markers.
5
Degenerate Primers for Insect PPO Genes
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Degenerate primers were designed according to PPO1 conserved amino acid sequences in GenBank with the following accession nos: Bombyx mori PPO (AAG09304), Choristoneura fumiferana PPO (ABW16859), Galleria mellonella PPO (AAK64363), Hyphantria cunea PPO (AAC34251), Manduca sexta PPO (AAC05796), and Spodoptera frugiperda PPO (ABB92834).
The forward primer was 5′-GGCSTACTTCCGCGARGACAT-3′ (where S = C or G and R = A or G) and the reverse primer was 5′-GCNGTCGCNGAGTCDCCCATCA-3′ (where N = A or C or G or T and G = A or G or T). PCR was carried out on T-Gradient Thermoblock (Biometra); the reaction conditions were as follows: 94°C with 4 minutes for denaturation, followed by 35 cycles at 94°C with 1 minute for denaturation, at 60°C with 1 minute for annealing, at 72°C with 1 minute for extension, and at 72°C with a final 10-minute extension. The obtained PCR products were tested by 1.5% agarose gel electrophoresis, purified with TIANgel Midi Purification Kit (Tiangen), ligated into the pMD18-T vector (TaKaRa), and transformed into competent cells of Escherichia coli strain DH5α. The positive clones were selected and sequenced on ABI PRISM 3730 by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd.
The forward primer was 5′-GGCSTACTTCCGCGARGACAT-3′ (where S = C or G and R = A or G) and the reverse primer was 5′-GCNGTCGCNGAGTCDCCCATCA-3′ (where N = A or C or G or T and G = A or G or T). PCR was carried out on T-Gradient Thermoblock (Biometra); the reaction conditions were as follows: 94°C with 4 minutes for denaturation, followed by 35 cycles at 94°C with 1 minute for denaturation, at 60°C with 1 minute for annealing, at 72°C with 1 minute for extension, and at 72°C with a final 10-minute extension. The obtained PCR products were tested by 1.5% agarose gel electrophoresis, purified with TIANgel Midi Purification Kit (Tiangen), ligated into the pMD18-T vector (TaKaRa), and transformed into competent cells of Escherichia coli strain DH5α. The positive clones were selected and sequenced on ABI PRISM 3730 by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd.
6
ChIP Assay for ERG Binding Sites
7
Optimizing Primer Annealing Temperatures
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In order to select the optimal annealing temperature for each primer set, PCR reactions were run using a temperature gradient ranging from 60 °C to 75 °C, with even temperature increments spanned across twelve thermocycler wells to identify the temperature at which a single specific product was produced with the highest yield, using a Biometra T-gradient thermoblock. PCR products were visualized by agarose gel electrophoresis. Primers were used to amplify their respective gene targets at their optimal annealing temperature using the following program: 1 cycle: 2 min at 94 °C; 35 cycles: 1 min at 94 °C, 30 s at the appropriate annealing temperature, 30 s at 72 °C; 1 cycle: 5 min at 72 °C using a BIORAD C1000 Touch thermal cycler. The PCR products were sequenced at the Center for Applied Genomics (Toronto Sick Kids Hospital), and sequences obtained were subjected to nucleotide Basic Local Alignment Search Tool (blastn) analysis (National Center for Biotechnology Information (NCBI)) to confirm PCR product identity and primer specificity.
8
Chromatin Immunoprecipitation for ERG Regulation
9
Quantitative Gene Expression Analysis
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RNA isolation was performed as previously described (30 (link)). Total RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. cDNA was synthesized from 1 mg of total RNA using reverse transcriptase (TaKaRa Biotechnology, Otsu, Japan). MMP-9 transcript expression levels were determined using the MiniOpticon Real-Time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). qPCR was performed in a thermocycler (Biometra, T-Gradient Thermoblock, Germany) with a reaction volume of 10 μl containing 0.03 μg complementary DNA product, 2 μM forward and reverse primers and the KAPA™ SYBR® FAST qPCR reagent (Kapa Biosystems, Wilmington, MA, USA). The primers used were as follows: Forward, 5′-GAACCAATCTCACCGACAGG-3′, and reverse, 5′-GCCACCCGAGTGTAACCATA-3′ for MMP-9; and forward, 5′-TCTGCTGGAAGGTGGACAGT-3′, and reverse, 5′-CCTCTATGCCAACACAGTGC-3′ for β-actin. Cycling conditions were as follows: 40 cycles of 95°C for 5 sec and 60°C for 34 sec. β-actin was included as a reference control. The comparative 2−ΔΔCt method was used to calculate the relative expression of each gene (30 (link)).
10
Quantitative RT-PCR Analysis of Murine Cytokines
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Quantitative real time-PCR was used to evaluate cultured spleen cells. Total RNAs from cultured spleen cells were extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the protocol provided by the manufacturer and quantified using a NanoDrop 200 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA (500 ng) was reverse transcribed to obtain complementary DNA (cDNA) using the ImProm-II™ Reverse Transcription system (Promega, Madison, WI, USA) following the manufacturer’s instructions in a T-Gradient Thermoblock (Biometra, Gottingen, Germany). All TaqMan® Gene expression primers and probes for murine IL-4, IL-5, IL-17A, IL-22, IFN-γ, and GAPDH were designed by Applied Biosystems (Foster City, CA, USA) (as Inventoried Assays). The assay ID details were as follows: IL-4 (Mm00445259_m1), IL-5 (Mm00439646_m1), IL-13 (Mm00434204_m1), IL-17A (Mm00439618_m1), IL-22 (Mm01226722_g1), IFN-γ (Mm01168134_m1), and GAPDH (Mm99999915_g1). qRT-PCR was performed using TaqMan® Universal PCR Master mix (Applied Biosystems) and the StepOnePlus™ Real-Time PCR system (Applied Biosystems). The transcript level for each gene was normalised to that of the internal control gene GAPDH. Relative gene expression was acquired using the ΔΔCt method, where Ct = threshold cycle value.
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