Another independent experiment was conducted in which the freshly detached shoot sample of 15-day-old Se-2-treated B. rapa plants was immediately stored in liquid nitrogen. For the extraction of total RNA, the TransZol Up reagent (TransGen Biotech, Beijing, China) was used. RNA quality and quantity were analyzed using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For complementary DNA (cDNA) production, 2 µg RNA was transferred into the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit according to the manufacturer’s instructions (TransGen Biotech, Beijing, China). Subsequently, a 1:10 dilution of cDNA was used for quantitative real-time PCR analysis with the TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) attached to a Roche LightCycler 480 thermal cycler instrument (384-well; Roche, Basel, Switzerland). The primers listed below were used to assess the expression levels of the APX, SOD, POD, and CAT genes (Zhang et al., 2020 (link)).
Lightcycler 480 thermal cycler
Manufactured by Roche
Sourced in Germany, Switzerland, Japan
The LightCycler 480 thermal cycler is a real-time PCR instrument designed for quantitative gene expression analysis and genotyping. It features a detection system that can measure fluorescence in up to 4 channels simultaneously. The device is capable of performing PCR reactions in 96- or 384-well microplates.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Qiaamp viral rna mini kit, by Qiagen (3 mentions)
Lightcycler 480 sybr green 1 master, by Roche (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 rna master hydrolysis probes kit, by Roche (2 mentions)
Trizol, by Merck Group (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Transzol up reagent, by Transgene (1 mentions)
Transscript one step gdna removal and cdna synthesis supermix kit, by Transgene (1 mentions)
Transstart tip green qpcr supermix, by Transgene (1 mentions)
Sybr green premix pro taq hs qpcr kit 2, by Accurate Biology (1 mentions)
Transzol up plus rna kit, by Transgene (1 mentions)
Evo m mlv rt kit with gdna clean for qpcr 2, by Accurate Biology (1 mentions)
Kod sybr qpcr kit, by Toyobo (1 mentions)
Sensifast cdna synthesis kit, by Meridian Bioscience (1 mentions)
Rt pcr kit, by Takara Bio (1 mentions)
24 protocols using lightcycler 480 thermal cycler
1
RNA extraction and qRT-PCR analysis
2
Comprehensive Intestinal Gene Expression Analysis
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The total RNA of the intestine was extracted by TransZol Up Plus RNA kits (TransGen, China), and the RNA concentration was determined by Spectrophotometric analysis (Nanodrop 2000). The cDNA was reverse-transcribed from total RNA by Evo M-MLV RT kit with gDNA Clean for qPCR II (Accurate Biotechnology Hunan Co., Ltd, China). The gene expressions of trypsin, amylase, lipase, superoxide dismutase (SOD), lipopolysaccharide (LPS) and beta-1,3-glucan binding protein (LGBP), prophenoloxidase (PPO), phenoloxidase (PO), Crustin (CRU), anti-lipopolysaccharide factor (ALF), penaeidin (PEN), and lysozyme (LYZ) were assessed by the Roche Light Cycler480 thermal cycler (Roche Applied Science, Germany) using the SYBR® Green Premix Pro Taq HS qPCR Kit II (Accurate Biotechnology Hunan Co., Ltd, China). For each target gene, specific primers were designed by Primer 5.0 software according to the known sequences in the NCBI database (Table 2
3
Quantitative RT-PCR for POFUT1 Expression
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Cells and tissues were treated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) for RNA extraction. cDNA synthesis and real-time quantitative PCR (q-PCR) were performed using the TOYOBO RT kit and KOD SYBR® q-PCR kit (TOYOBO Co., Ltd, Osaka, Japan). Primers used in the study were as follows: POFUT1: 5′-AACCAGGCCGATCACTTCTTG-3′ (Forward), and 5′-GTTGGTGAAAGGAGGCTTGTG-3′ (Reverse). GAPDH: 5′-GCACCGTCAAGGCTGAGAAC-3′ (Forward), and 5′-TGGTGAAGACGCCAGTGGA-3′ (Reverse). Reactions were performed according to the manufacturer’s protocols using the Roche LightCycler 480 thermal cycler (Roche, Switzerland).
4
Immune Response Dynamics in L. vannamei
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Eleven tissues, including the hemocytes, hepatopancreas, gill, heart, stomach, pyloric caecum, nerve, epithelium, eyestalk, intestine and muscle, were obtained from 10 healthy L. vannamei for RNA extraction. For the challenge experiments, 60 healthy L. vannamei divided into two groups (n = 30) were intramuscularly injected with WSSV (106 copies/g) in the third abdominal segment. L. vannamei injected with PBS were used as controls. At 0, 4, 8, 12, 24, 48, and 72 h post-injection (hpi), three animals from each group were randomly sampled for hemocyte, hepatopancreas, gill and intestine collection. Total RNA extraction, reverse transcription and real-time PCR (qPCR) analysis were performed as detailed in previous research (30 (link)). The qPCR was performed in a Roche Light Cycler480 thermal cycler (Roche Applied Science, Germany). Elongation factor 1α (EF1α, GenBank accession No. GU136229) was used as the internal control. The expression level of LvGSK3β was calculated using the Livak(2−ΔΔCt) method after normalization to LvEF1α. All samples were tested in triplicate. Primer sequences are listed in Table 1
5
Colon Tissue RNA Extraction and qPCR Analysis
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Total RNA was extracted from the proximal colon tissues by homogenizing in Trizol reagent (Invitrogen) using a Polytron(IKA®ULTRATURRAX®). cDNA synthesis was performed by using SensiFASTTM cDNA Synthesis Kit (Bioline, UK) according to the manufacturer’s protocol. qPCR was performed by using SensiFASTTM SYBR No-ROX kit master mix (Bioline, UK) in Roche LightCycler480 Thermalcycler (Roche Life Science). All expression levels were normalized to that of endogenous control glyceraldehyde-3-phosphate-dehydrogenase (gapdh) and results were analysed using the comparative threshold method. For primer and RT-PCR protocol details, see supplementary Table S2 .
6
POFUT1 Gene Expression Quantification
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POFUT1 messenger RNA (mRNA) expression was detected in GC cells using quantitative real-time polymerase chain reaction (q-PCR). Cells were treated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for RNA extraction. Complementary DNA (cDNA) was synthesized using an RT-PCR kit (TaKaRa, Tokyo, Japan) and amplified using q-PCR, according to the protocol of the SYBR q-PCR Kit (Toyobo, Osaka, Japan) using a Roche LightCycler 480 thermal cycler (Roche, Basel, Switzerland). The primers used were as follows: POFUT1: 5′-AACCAGGCCGATCACTTCTTG-3′ (forward); 5′-GTTGGTGAAAGGAGGCTTGTG-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5′-GGAGCGAGATCCCTCCAAAAT-3′ (forward); 5′-GGCTGTTGTCATACTTCTCATGG-3′ (reverse). Gene transcripts were normalized to GAPDH and the relative fold change was calculated using the 2 -ΔΔCT method.
7
Quantifying EBOV and MARV RNA Levels
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FILOcep1&2- and control eGFP-vaccinated mice were challenged with either mouse-adapted EBOV or MARV. Blood and tissues (liver, spleen, kidney and lungs) from 4 mice per vaccinated group were collected upon euthanasia at day 3 and 5 post-infection to determine viral RNA levels. RNA of mouse blood and tissues were extracted using QIAamp viral RNA minikit (Qiagen) and the RNeasy mini Kit (Qiagen) according to the manufacturer's instructions. Viral RNA levels were quantified by reverse transcription quantitative PCR (RT-qPCR) targeting viral polymerase gene and using the Light Cycler 480 thermal cycler (Roche, Germany). The primers and probes are shown in Table 3 . Cycling conditions were as follows: 63°C for 3 min and 95°C for 30 sec, followed by 45 cycles of 95°C for 15 sec and 60°C for 30 sec.
8
Quantifying HIV and Marburg Virus RNA
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For HIV-based infections, total RNA was extracted from infected cells by use of an RNA extraction kit (Tiandz, Beijing, China). The level of viral RNA was determined by performing qRT-PCR analysis by use of a one-step SYBR PrimeScript RT-PCR kit (TaKaRa, Dalian, China). The primer pair (5′-TTAAGCCTCAATAAAGCTTGCC-3′ and 5′-GTTCGGGCGCCACTGCTAGA-3′) amplifies the long-terminal repeats of HIV. Levels of cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA were amplified with primers 5′-ATCATCCCTGCCTCTACTGG-3′ and 5′-GTCAGGTCCACCACTGACAC-3′; the results served as an internal control to normalize the level of HIV RNA.
For MARV infections, viral RNA was extracted from cell culture supernatants using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). Viral RNA levels were quantified by qRT-PCR using the LightCycler 480 thermal cycler (Roche, Germany) and the LightCycler 480 RNA Master Hydrolysis Probes kit (Roche, Germany) along with the following primers and probe:
1-F, 5′-GCAAAAGCATTCCCTAGTAACATGA-3′; 1-R, 5′-CACCCCTCACTATRGCGTTYTC-3′; 2-F, 5′-GCGAAGGCATTCCCTAGTAATATGA-3′;
2-R, 5′-CACCTCTTACTATGGCATTCTC-3′; probe, 5′-56-FAM/TGGCACCAY/ZEN/AATTCAGCAAGCATAGG/3IABkFQ-3′.
Cycling conditions were as follows: 63 °C for 3 min and 95 °C for 30 s, then 45 cycles of 95 °C for 15 s and 60 °C for 30 s.
For MARV infections, viral RNA was extracted from cell culture supernatants using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). Viral RNA levels were quantified by qRT-PCR using the LightCycler 480 thermal cycler (Roche, Germany) and the LightCycler 480 RNA Master Hydrolysis Probes kit (Roche, Germany) along with the following primers and probe:
1-F, 5′-GCAAAAGCATTCCCTAGTAACATGA-3′; 1-R, 5′-CACCCCTCACTATRGCGTTYTC-3′; 2-F, 5′-GCGAAGGCATTCCCTAGTAATATGA-3′;
2-R, 5′-CACCTCTTACTATGGCATTCTC-3′; probe, 5′-56-FAM/TGGCACCAY/ZEN/AATTCAGCAAGCATAGG/3IABkFQ-3′.
Cycling conditions were as follows: 63 °C for 3 min and 95 °C for 30 s, then 45 cycles of 95 °C for 15 s and 60 °C for 30 s.
9
Quantifying Stem Cell Differentiation
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Cell lysis and RNA extraction were performed using RNeasy mini-kit by Qiagen (Hilden, Germany). The M-MLV RT (Moloney Murine Leukemia Virus Reverse Transcriptase) enzyme (provided by Promega™) was used for cDNA-synthesis. qPCR was measured in a Roche Lightcycler® 480 thermal-cycler. FAM-MGB-conjugated TaqMan Probes (by Thermo Fisher Scientific, Waltham, MA, USA) and GAPDH (assay ID: Hs99999905_m1, amplicon length: 122 bp) as a housekeeping control were used. For osteogenic differentiation, ALPL gene (assay ID: Hs01029144_m1) and BGLAP gene (assay ID: Hs01587814_g1) were measured at amplicon lengths of 79 and 138 bp, respectively. For adiopgenic differentiation, PPARG gene (assay ID: Hs01115513_m1) and CEBPA gene (assay ID: Hs00269972_s1) were measured at amplicon lengths of 90 and 77 bp, respectively.
10
Quantitative Real-Time PCR Analysis of Arabidopsis
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For quantitative real-time PCR analyses, Arabidopsis seedlings of the indicated genotypes were grown for 5 days as indicated above for naturally initiated LRP. For each genotype, 10 seedlings were pooled together and ground in liquid nitrogen. RNA extraction on these ground samples was performed using RNeasy Plant Kit (QIAGEN), including ''on column DNase treatment,'' following manufacturer's instruction. RNA concentration was measured with Nanodrop and all samples were diluted to 100 ng/mL. 500 ng of total RNA were used for cDNA synthesis using QuantiTect Reverse Transcription Kit (QIAGEN), including gDNase treatment (for the second time) prior to cDNA synthesis. cDNA samples were diluted 25 times and 5 mL were used as template in a 20 mL total reaction volume, using LightCycler 480 SYBR Green 1 Master (Roche Life Science) according to the manufacturer's instructions. Each sample was loaded in duplicate into a 96-well qPCR plate (Roche Life Science) and qPCR was run in a LightCycler 480 thermal cycler (Roche Life Science) for 45 cycles (initial denaturation at 98 C for 3 min, cycling: denaturation at 95 C for 5 s, annealing at 55 C for 10 s, elongation at 72 C for 30 s; melting curve from 45 C to 95 C with acquisitions every 1 C).
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