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Sybr green premix pro taq hs qpcr kit

Manufactured by Accurate Biology
Sourced in China, United States, Switzerland, Germany, Japan

The SYBR Green Premix Pro Taq HS qPCR Kit is a reagent designed for real-time quantitative PCR (qPCR) experiments. It contains a premixed solution that includes SYBR Green I dye, a hot-start DNA polymerase, and necessary buffers and reagents for the qPCR reaction.

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389 protocols using sybr green premix pro taq hs qpcr kit

1

Liver Gene Expression Analysis Protocol

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The total RNA of liver samples was extracted using the AG RNAex Pro reagent (Accurate Biology, Hunan, China). The cDNA was acquired by the reverse transcription (RT) kit (Accurate Biology, Hunan, China). The cDNA samples were amplified by real-time quantitative polymerase chain reaction with SYBR® Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology, Dalian, China). The mRNA expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB), NLRP3, sirtuin1 (Sirt1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme-oxygenase 1 (HO-1), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase (CAT), glutathione peroxidase 1 (GPX1), NAD(P)H quinone oxidoreductase-1 (NQO1), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) in the liver samples were assessed using a LightCycler 96 (Roch, Switzerland) with SYBR® Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology, Da Lian, China). All the primer sequences are listed in Table 2. β-actin was used as an internal reference gene, and the 2−ΔΔCT method was used to calculate the messenger ribonucleic acid (mRNA) expression of target genes relative to β-actin (17 (link)).
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2

Quantitative RT-PCR for Vibrio alginolyticus

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qRT-PCR was performed as previously described (67 (link), 68 (link)). Total RNA was extracted from V. alginolyticus with TRIzol reagent (Invitrogen, USA). The quality and the purity of RNA were checked by electrophoresis and Nanodrop. Reverse transcription was performed with EvoM-MLV RT kit with gDNA clean for qPCR (AG11705; Accurate Biology). The reaction for qRT-PCR was conducted in a 10-μl reaction including 5 μl 2× SYBR green premix pro Taq HS qPCR kit (AG11701; Accurate Biology), 2.6 μl H2O, 2 μl cDNA template, and 0.2 μl forward and reverse primers. Three technical replicas were included for each sample, and the reaction was run on a CFX384 Touch (Bio-Rad, USA).
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3

Quantitative RT-PCR Analysis of GBS

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qRT-PCR was performed as previously described [19 (link),66 (link)]. Total RNA was isolated from GBS with TRIzol (Invitrogen Life Technologies, United States) after treatment with lysozyme for 30 min at 37℃. The quality of RNA was monitored by 1% electrophoresis and Nanodrop. Reverse transcription was conducted with 1 μg total RNA with EvoM-MLV RT kit with gDNA clean for qPCR (AG11705; Accurate Biology). And qRT-PCR was performed in a reaction volume of 10 μl including 5 μl 2×SYBR green premix pro Taq HS qPCR kit (AG11701; Accurate Biotechnology), 2.6 μl H2O, 2 μl cDNA template, and 0.2 μl each of forward and reverse primers (10 mM) in 384-well plates. Primers used in this study was included in Supplementary Table S1. The reactions were set on a LightCycler 480 system (Roche, Germany). The cycling parameters were 95°C for 30 s, 40 cycles of 95°C for 10 s, and 60°C for 30 s. Fluorescence were measured at 72°C for 1 s during each cycle. At last, reaction was terminated at 95°C with a calefactive velocity of 5°C/s to obtain the melting curve. Data are shown as the relative mRNA expression compared with control group and standardized with the endogenous reference 16S rRNA gene. Statistic significance was calculated by 2−ΔΔCt method [67 (link)].
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4

Quantitative RT-PCR Methodology

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qRT-PCR was carried out as described previously (64 (link), 65 (link)). Total RNA of each sample was isolated with TRIzol (Invitrogen, USA). The RNA was then quantified spectrophotometrically. qRT-PCR was carried out on 1 μg of total RNA by using an EvoM-MLV RT kit with gDNA clean for qPCR (AG11705; Accurate Biotechnology) according to the manufacturer’s instructions. The cDNAs were synthesized with a reverse transcription kit and used as the template for qRT-PCR. qRT-PCR was performed in 384-well plates with a total volume of 10 μl containing 5 μl 2× SYBR green premix pro Taq HS qPCR kit (AG11701; Accurate Biotechnology), 2.6 μl H2O, 2 μl cDNA template, and 0.2 μl each of forward and reverse primers (10 μM). All samples were tested in biological triplicate and run on a CFX384 Touch (Bio-Rad, USA) according to the manufacturer’s instructions. The cycling parameters were 95°C for 30 s to activate the polymerase; 40 cycles of 95°C for 10 s; and 56°C for 30 s. Fluorescence measurements were performed at 72°C for 1 s during each cycle. Cycling was terminated at 95°C with a calefactive velocity of 5°C/s to obtain a melting curve. The expression of samples was normalized by β-actin, gapdh, and ef1α using the 2−ΔΔCT method (66 (link)). All the primers are listed in Table S1B.
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5

Quantitative PCR Reference Gene Analysis

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The basic transcription factor 3 (BTF3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were selected as reference genes to perform CNV detection and mRNA-level examination [32 (link)]. The LightCycler® 96 Instrument (Roche, Basel, Switzerland) was used in our research. The 20 μL reaction mixtures contained 10 μL SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Hunan, China), 6.8 μL ddH2O, 0.8 μL of upstream and downstream primers, and 1.6 μL of DNA/cDNA. The qPCR conditions were 60 s at 95 °C, and then 45 cycles of 10 s at 95 °C, and then 30 s at 60 °C. Subsequently, the melting curve was 30 s at 95 °C, and 0.02 s at 55 °C, and then 0.5 °C per cycle to 95 °C. Analysis of each DNA and RNA sample was replicated three times, and the experimental results were represented by mean values ± standard error (SE).
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6

RNA Extraction and qPCR Analysis

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Total RNA was extracted using TRIzol reagent (Accurate Biotechnology, Hunan, China) and converted into cDNA (Evo M-MLV RT Premix for qPCR; Accurate Biotechnology). The miRNA RNAs were converted into cDNA using a miRNA 1st strand cDNA synthesis kit (Accurate Biotechnology, Hunan, China). β-Actin and U6 were used as reference controls for mRNAs, lncRNAs, and miRNAs. Primers were designed by Accurate Biotechnology (Hunan, China) (Table S2). qPCR was performed using the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China) and the Applied Biosystems 7500 Real-Time PCR Detection System. The data were analyzed using the 2ΔΔCt (Livak) relative expression method. All experiments were repeated three times.
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7

Quantifying Gene Expression in Intestinal Mucosa

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Total RNA was isolated from the liquid-nitrogen-pulverized jejunal and ileal mucosa samples with the RNAiso Plus reagent (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s protocol. The concentration and quality of the RNA were determined using the NanoDrop One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728, Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) following the manufacturer’s instructions. The RT-qPCR was then performed on a LightCycler R480II Real-Time PCR Instrument (Roche, Basel, Switzerland) using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) in accordance with manufacturer’s instructions. The fluorescence PCR program was set as follows: pre-denaturation, 95 °C for 30 s, 1 cycle; PCR amplification, 95 °C for 5 s, 60 °C for 30 s, 40 cycles; melting, 95 °C for 5 s, 60 °C for 1 min, 1 cycle; cooling, 50 °C for 30 s. Primers used in the PCR assay are listed in Table 1. The relative expression of selected genes normalized by β-actin was calculated using the 2−ΔΔCt method [22 (link)]. The data were expressed as relative values to those for the CON-NBW group.
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8

RNA Extraction and qRT-PCR Analysis

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The total RNA was extracted from samples using TRIzol and reverse-transcribed using the Evo M-MLV RT Kit with gDNA Clean for qPCRII (AG11711, Accurate Biotechnology, Changsha, China) according to the manufacturer’s protocol. qRT-PCR reactions were carried out using SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology, Changsha, China) and QuantStudio 1 (Appliedbiosystems by Thermo Fisher Scientific, Shanghai, China). The relative expression was calculated via the 2−ΔΔCt method [65 (link)], and qRT-PCR primer sequences are listed in Supplementary Table S8.
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9

Real-time qPCR for gene expression analysis

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Total RNA was extracted from cultured cells using a commercially available kit (Qiagen, #74136) with the optional DNase digestion step. The RNA was reverse transcribed with an Evo M-MLV RT kit with gDNA Clean for qPCR (Accurate Biotechnology, China) and qRT-PCR was performed using a Roche LightCycler 480 with a SYBR Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology) and the following cycling conditions: 95°C for 5 min followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The relative level of each target gene was normalized to that of endogenous Rpl19 and calculated using the comparative Ct (ΔΔCt) method. The sequences of the utilized primers were as follows: 5′- TGGAAAATGGTTCGAGAGTCAG-3′ (forward) and 5′- CATTCCGTCTCTAGGTTAAAGCG-3′ (reverse) for Opa1; 5′-ACGGAGGCTGGGATGCCTTTG-3′ (forward) and 5′-AGTGATGCAGGCCCCGACCA-3′ (reverse) for Bcl2; and 5′-ACCTGGATGAGAAGGATGAG-3′ (forward) and 5′-ACCTTCAGGTACAGGCTGTG-3′ (reverse) for Rpl19.
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10

Comprehensive qPCR Analysis of Immune Biomarkers

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Total RNA was extracted using the RNAex Pro Reagent (Accurate Biotechnology) according to the manufacturer’s instructions. In addition, RNA was reverse-transcribed according to the mRNA Reverse Transcription Kit (Accurate Biotechnology); qPCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) on a 7500 RT PCR System (Applied Biosystems, Foster City, CA, USA). GAPDH was selected as an internal control, and all qPCR reactions were performed in triplicate. The relative mRNA expression level was calculated using the 2−ΔΔCt method. The specific primers were listed in Table 1.

Specific primers

GenePrimer sequence (5′ to 3′)Size (bp)
EZH2F:GGACGAAGAATAATCATGGGCC116
R:CGTCTGAACCTCTTGAGCTGTCT
GAPDHF:AGAAGGCTGGGGCTCATTTG258
R:AGGGGCCATCCACAGTCTTC
IL-10F:TCAAGGCGCATGTGAACTCC176
R:GATGTCAAACTCACTCATGGCT
TGF-βF:GACTCGCCAGAGTGGTTATCT154
R:CGGTAGTGAACCCGTTGAT
IL-1βF:AGCTACGAATCTCCGACCAC186
R:CGTTATCCCATGTGTCGAAGAA
TNF-αF: TCTCGAACCCCGAGTGACAA181
R: TGAAGAGGACCTGGGAGTAG
IL-6F:AATAACCACCCCTGACCCAAC149
R:ACATTTGCCGAAGAGCCCT
IL-4F:CGGCAACTTTGTCCACGGA111
R:TCTGTTACGGTCAACTCGGTG
CXCL-16F:GACATGCTTACTCGGGGATTG170
R:GGACAGTGATCCTACTGGGAG
PD-L1F:GGACAAGCAGTGACCATCAAG235
R:CCCAGAATTACCAAGTGAGTCCT
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