Total RNA was extracted using RNAiso Plus (TaKaRa) kit and reverse-transcribed into cDNA by RevertAid First Strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s instructions. Quantitative PCR analysis was performed using iTaq Universal SYBR Green supermix (2×) (BIO-RAD). The primer pairs used for quantitative PCR are: PBK (human) forward primer: 5′-CCTTTGGCCTTACTTTGTG -3′; PBK (human) reverse primer: 5′-ACGATCTTTAGGGTCTTCAT-3′; GAPDH (human) forward primer: 5′-AACGGATTTGGTCGTATTG-3′; GAPDH (human) reverse primer: 5′- GGAAGATGGTGATGGGGAT -3′.
Itaq universal sybr green supermix 2
Manufactured by Bio-Rad
Sourced in United States
ITaq Universal SYBR Green Supermix (2×) is a ready-to-use, 2X concentrated, thermostable DNA polymerase-based master mix that contains SYBR Green I dye for real-time PCR applications. The supermix includes all the necessary components for PCR amplification and detection, except primers and template DNA.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Bio-Rad (2 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Quick rna isolation kit, by Huayueyang (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Cfx96 thermocycler, by Bio-Rad (1 mentions)
Spss v16, by IBM (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Cfx384 real time pcr detection system, by Bio-Rad (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Quantitect rev transcription kit, by Qiagen (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Pcr primer design tool, by Eurofins (1 mentions)
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using itaq universal sybr green supermix 2
1
Quantitative Analysis of PBK Expression
2
Quantitative Expression Analysis of Tea Transcripts
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Total RNA was isolated from finely powdered tea leaves (100 mg) using the Quick RNA Isolation Kit (Huayueyang, Beijing, China). First-strand cDNA was synthesized from total RNA (500 ng). The qRT-PCR system contained iTaq Universal SYBR Green Supermix (2×) (5 µl; Bio-Rad, Hercules, CA, USA) and 0.4 µM of each specific forward and reverse primer. The primers used are listed in Supplementary Table S1 at JXB online. Elongation factor 1-α (CsEF genes) was used as an internal reference. qRT-PCR was conducted on a Roche LightCycler 480 system (Roche Applied Science, Mannheim, Germany) under the following conditions: one cycle of 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. A melt curve was performed at the end of each reaction to verify PCR product specificity. The 2−Δct method was used to calculate relative expression levels (Livak and Schmittgen, 2001 (link)).
3
Genotypic Spa Typing of S. aureus
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The 18 strains of S. aureus were submitted to genotypic spa typing by RT-PCR using the primers spa1113F and spa1514R F [15 (link)]. The amplification reactions of the DNA were performed on a CFX96 thermocycler (BioRad Inc., Hercules, FL, USA) with a total volume of 50 μL per reaction containing 5 μL of DNA from each strain, 200 nM forward primer spa1113F (5’-TAAAGACGATCCTTCGGTGAGC-3’) (STAB VIDA Lda., Lisboa, Portugal), 200 nM reverse primer 1492R (5’-CAGCAGTAGTGCCGTTTGCTT-3’) (STAB VIDA Lda., Lisboa, Portugal) and 1x iTaq™ Universal SYBR® Green Supermix (2×) (BioRad Inc., Hercules, USA). The amplification was conducted under the following conditions: initial denaturation at 95 °C for 5 min, followed by denaturation for 35 cycles at 95 °C for 15 s, annealing at 60 °C for 45 s and extension at 72 °C for 90 s.
The amplified products were purified (QIAquick PCR Purification Kit, Hilden, Germany) and Sanger-sequenced at STAB VIDA Lda. (Lisboa, Portugal). The resulting chromatograms were analysed using the UGENE program (version 38.1, Unipro, Novosibirsk, Russia) for the extraction of high-quality fragments, which were further processed using DNAGear software [16 (link)].
The amplified products were purified (QIAquick PCR Purification Kit, Hilden, Germany) and Sanger-sequenced at STAB VIDA Lda. (Lisboa, Portugal). The resulting chromatograms were analysed using the UGENE program (version 38.1, Unipro, Novosibirsk, Russia) for the extraction of high-quality fragments, which were further processed using DNAGear software [16 (link)].
4
Validating Unigenes Expression via qRT-PCR
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Sixteen unigenes (10 unigenes for original transcriptome verification and six unigenes for commercial insecticide verification) were selected and validated by quantitative real-time PCR (qRT-PCR) on a CFX-96 Real-Time PCR Detection System (Bio-Rad, Berkeley, CA, USA) with specific primers designed by Primer Premier 5.0 (Table S1 ). The cDNA template used for RT-qPCR was reverse-transcribed from qualified total RNA using the Prime Script RT Reagent Kit (Bio-Rad, Berkeley, CA, USA). A 20 µL PCR reaction volume contained 10 µL of Bio-Rad iTaq™ Universal SYBR® Green Super mix (2 ×), 1 µL of each gene-specific primer (10 µM), 2 µL of each cDNA template and 6 µL of ddH2O. Actin was used as a reference gene and the 2−ΔΔCt method was employed to estimate relative changes in gene expression (Livak & Schmittgen, 2001 (link); Chang et al., 2017a (link)). Each treatment included four replicates, and each sample was assessed in triplicate. Significant differences among treatments were detected by one-way ANOVA, followed by Tukey’s multiple comparison and analysis with SPSS v. 16.0 (SPSS, Chicago, IL, USA); significant differences were determined at P < 0.05.
5
Quantitative Analysis of FGFR and FGF20 Expression
6
Quantitative Real-time RT-PCR Analysis
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Real-time quantitative cDNA was synthesized using instructions provided with the PrimeScript RT Reagent Kit (Bio-Rad, Berkeley, CA, USA), and primers were designed using Primer 5.0 software (Table 1 ). Reactions were conducted using a CFX-96 Real-Time PCR System (Bio-Rad, Berkeley, CA, USA), and each PCR reaction included three replicates. PCR reactions included iTaq Universal SYBR Green Supermix (2×) (Bio-Rad, Berkeley, CA, USA), one μL of each forward and reverse primer (10 μmol L–1) (Table 1 ), two μL of cDNA template (2.5 × 10–4 μg μL–1), and six μL of ddH2O. Quantitative real-time reverse transcriptase PCR conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 30 s, and 56.3 °C for 15 s; melting curve analysis was then performed to determine the specificity of PCR products.
7
qRT-PCR Assay for L. trifolii
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RNA (0.5 μg) was reverse-transcribed into first-strand cDNA using the Bio-Rad iScript™ cDNA Synthesis Kit (Bio-Rad, CA, USA). Reactions were conducted in a 20 μl reaction volume consisting of 10 μl Bio-Rad iTaq™ Universal SYBR® Green Supermix (2×), 1 μl of each gene-specific primer (10 μM) (Table 1 ), 2 μl of each cDNA template, and 6 μl of ddH2O. Real-time PCR were performed using an Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific, USA) under the following conditions: 3 min at 95°C, 40 cycles of denaturation at 95°C for 30 s, and annealing at the Tm of primer pairs (Table 1 ) for 30 s. Each treatment contained four replications, and each reaction was run in triplicate. β-actin was cloned from L. trifolii (GenBank accession no: KY231150) and used as a reference gene.
8
Quantifying Lactobacillus Functional Genes
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Genomic DNA was extracted from cell pellets collected from bioreactor fermentations with the GeneJET Genomic DNA Purification Kit following the protocol for DNA extraction from Gram‐positive microbes (ThermoScientific). The targeted genes encoded the rhamnose isomerase (rha) of L. rhamnosus, and the large subunit of the glycerol/diol dehydratase (pduC) of L. reuteri and L. coryniformis (Table 2 ). Primer pair rha1‐iso was designed based on L‐rhamnose isomerase gene sequence (WP_010011911.1) using PCR Primer Design Tool (Eurofins). To verify specificity of rha1‐iso, PCR was performed using DNA from L. rhamnosus FMT 736 as template.
Standards were prepared from 10‐fold dilution series of linearized plasmids harbouring gdh of L. reuteri, or the purified (Monarch PCR & DNA Cleanup Kit, New England Biolabs) and quantified (Qubit 4 fluorometer, Thermo Fisher Scientific) PCR product of rha‐iso. qPCR reactions were run in 96‐well plates with iTaq Universal SYBR Green Supermix 2× on a CFX Connect Real‐Time PCR Detection System (both Bio‐Rad). The program started with denaturation at 95°C for 3 min, and was followed by 40 cycles with each cycle having a denaturation step (95°C for 10 s), and annealing step (60°C for 30 s) followed by melting curve analysis. All samples were run as technical duplicates and each run included a negative control.
Standards were prepared from 10‐fold dilution series of linearized plasmids harbouring gdh of L. reuteri, or the purified (Monarch PCR & DNA Cleanup Kit, New England Biolabs) and quantified (Qubit 4 fluorometer, Thermo Fisher Scientific) PCR product of rha‐iso. qPCR reactions were run in 96‐well plates with iTaq Universal SYBR Green Supermix 2× on a CFX Connect Real‐Time PCR Detection System (both Bio‐Rad). The program started with denaturation at 95°C for 3 min, and was followed by 40 cycles with each cycle having a denaturation step (95°C for 10 s), and annealing step (60°C for 30 s) followed by melting curve analysis. All samples were run as technical duplicates and each run included a negative control.
9
Antioxidant and Immune Gene Expression in Laying Hens
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The liver tissues were randomly selected from three laying hens per replicate. The liver tissues were immediately removed and frozen at −20 °C until RNA extraction. Fifty milligrams of samples were mixed with lysis buffer and homogenized using a tissue homogenizer. RNA extraction was then carried out according to manufacturer’s protocol using a column-based RNA extraction kit (Invitrogen, PureLinkTM RNA Mini Kit, MA USA). The concentration and purity of total RNA was measured at an absorbance ratio of 260–280 nm using NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthesized using Bio-Rad iScriptTM RT Supermix cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The primers required for IL-1β, IL-6, IL-10, TNF-α, SOD, CAT, GSH-Px1, and Nrf2 genes were designed according to Table 2 . Employing the CFX ConnectTM Real-Time PCR System (Bio-Rad, USA) and the iTaq Universal SYBR Green supermix 2× (Bio-Rad, USA) in addition to the specific primers for individual genes, the qPCR reaction was conducted. The expression levels of the antioxidant and immune-related gene were measured using the 2−ΔΔCt method and a standard curve [23 (link)].
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