The RNA concentration and integrity (RIN score) were determined using the Agilent 2100 Bioanalyzer Pico-chips (Agilent Technologies) following manufacturing protocol. RNA libraries were prepared with poly(A) selection using 3′-SMART CDS Primer II A within the SMARTer Ultra Low Input RNA Kit for Sequencing–v3 (Clontech Takara) following manufacturer’s protocol including ERCC RNA Spike-In Mix (Life Technologies). Following first strand synthesis, cDNA was purified with 1× Agencourt AMPure XP beads (Beckman Coulter), as described in SMARTer protocol. Nextera® XT DNA Library Preparation kit (Illumina) was used for sample barcoding and fragmentation according to the manufacturer’s protocol. Library amplification and library barcoding were achieved within 12 cycles of PCR. Subsequent PCR products were purified with 1.8× Agencourt AMPure XP beads according Nextera XT protocol. Library quantification was done using the SYBR® FAST Universal qPCR Kit (KAPA Biosystems). Equal molar individual libraries were pooled, and the pool concentration was determined using the KAPA SYBR® FAST Universal qPCR Kit. Finally, libraries were diluted and denatured with the addition of 1% PhiX Sequencing Control V3 (Illumina). 75-bp paired-end reads were generated using NextSeq 500/550 High Output v2 kit (150 cycles) on an Illumina NextSeq (Illumina).
Sybr fast universal qpcr kit
Manufactured by Roche
Sourced in United States, Switzerland
The SYBR FAST Universal qPCR kit is a reagent system designed for real-time quantitative PCR (qPCR) analysis. It includes all necessary components for amplification and detection of DNA targets using the SYBR Green fluorescent dye. The kit provides a fast, sensitive, and versatile solution for gene expression analysis and quantification of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Agencourt ampure xp bead, by Beckman Coulter (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Quantstudio 6 flex system, by Thermo Fisher Scientific (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Goscript reverse transcription system, by Promega (3 mentions)
M mlv transcriptase, by Promega (3 mentions)
Lc480 real time pcr detection system, by Bio-Rad (3 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Agilent 2100 bioanalyzer pico chip, by Agilent Technologies (2 mentions)
Ercc rna spike in mix, by Thermo Fisher Scientific (2 mentions)
Nextseq, by Illumina (2 mentions)
Phix sequencing control v3, by Illumina (2 mentions)
Smarter ultra low input rna kit for sequencing v3, by Takara Bio (2 mentions)
Iq5 real time system, by Bio-Rad (2 mentions)
Premix pcr solution, by Takara Bio (2 mentions)
Lightcycler 480 system, by Roche (2 mentions)
Sybr fast universal qpcr mix, by Roche (2 mentions)
43 protocols using sybr fast universal qpcr kit
1
Transcriptome Profiling via RNA-Seq
2
Gene Expression Analysis of Antiviral Pathways
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RNA was extracted with the RNeasy mini kit (Qiagen) per the manufacturer’s protocol High-Capacity cDNA Reverse Transcription Kit was used to make cDNA. Quantitative reverse transcription PCR (qRT-PCR) was performed using an SYBR FAST universal qPCR kit (KAPA Biosystems). Predesigned KiCqStart primers for DDX58, IL6, IFITM3, IRF7, IFIH1, IFNA6, IFNG and HPRT1 were purchased from Sigma.
3
miRNA Expression Profiling in BMDMs
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Total RNA samples (both tissues and cells) were isolated from mice using TRIzol reagent (Ambion, Inc., Austin, Texas, US) per manufacturer’s protocol. We determined miRNA expression profiles of BMDMs treated with 10 nM PDX using miRNA microarray analysis. We reverse-transcribed the complementary deoxyribonucleic acid (cDNA) of mRNA (1 μg) and of the miRNA itself using a PrimeScript RT Reagent Kit (TaKaRa Bio, Shiga, Japan) and measured the expression of the target gene using a SYBR Fast Universal qPCR Kit (Kapa Biosystems, Inc. [Roche Life Science, Basel, Switzerland]) for RT-qPCR. Primer sequences used are listed in online Supp. Table S2 . We normalized miRNAs to U6 controls and mRNAs to β-actin; conversion was performed using relative quantification (2–ΔΔCt).
4
Quantitative Analysis of SOD2 Expression
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Reverse transcription-PCR (RT-PCR) was performed by amplifying complementary DNA (cDNA) in Premix PCR solution (Takara, Shiga, Japan) with SOD2 primers (5′-GGA-AGC-CAT-CAA-ACG-TGA-CTT-3′ and 5′-GTG-CTC-CCA-CAC-ATC-AAT-CC-3′). Quantitative real-time PCR was performed using the SYBR Fast Universal qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) and SOD2 primers (5′-GGC-CTA-CGT-GAA-CAA-CCT-GAA-3′ and 5′-CTG-TAA-CAT-CTC-CCT-TGG-CCA-3′). GAPDH was amplified in both PCR assays with the following primers as an internal control for normalization: 5′-CAT-CTC-TGC-CCC-CTC-TGC-TGA-3′ and 5′-GGA-TGA-CCT-TGC-CCA-CAG-CCT-3′. The RT-PCR and real-time PCR results were analyzed by agarose gel electrophoresis and an IQ-5 Real-Time System (Bio-Rad), respectively.
5
Quantification of mRNA Expression in HepG2 Cells
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HepG2 cells were plated at a density of 3 × 105 cells/well in a six-well plate and incubated overnight, before being starved for 12 h in DMEM with 0.5% BSA and used for different treatments as described above; one group was treated with QCT (10 μM) as a comparison. Total RNA from HepG2 cells was extracted using an RNAprep pure Cell/Bacteria Kit (Tiangen Biotech Co., Beijing, China). The purity and concentration of RNA were determined with a Genova Nano spectrophotometer (BIBBY JENWAY, Staffordshire, UK). Complementary DNA (cDNA) was synthesized with a FastQuant RT Kit (With gDNase) (Tiangen Biotech Co., Beijing, China) according to the manufacturer’s protocols. The relative levels of mRNA to GAPDH were analyzed using an SYBR fast universal qPCR kit (KAPA Biosystems, MA, USA) and specific primers. The primer sequences are shown in Table 2 . The qRT-PCR was performed on an ABI Quant 5 PCR system using the 2−ΔΔCt method. GAPDH was used as the normalized reference gene.
6
RT-qPCR Quantification of DENV Gene Expression
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The SYBR Green dye binding system was used for RT-qPCR in this study. SYBR Green binds the minor groove of DNA, and the target gene expression was quantified by detecting the resulting fluorescence signal. The cDNA sample was quantified using a KAPA SYBR FAST Universal qPCR kit (KAPA), via the qPCR primers (S7: 5′-TCAGTGTACAAGAAGCTGACCGGA-3′/5′-TTCCGCGCGCGCTCACTTATTAGATT-3′; DENV:5′-GAAGA CATTGACTGYTGGTGCAA-3′/5′-CGATGTTTCCACGCCCC TTC-3′). The PCR protocol consisted of initial denaturation at 95°C for 3 min, followed by 40 cycles of 3 s at 94°C and 40 s at 60°C. Fluorescence readings were measured at 72°C after each cycle. The target gene signal was detected and analyzed using the ABI 7900HT Fast Real-Time PCR System, and relative quantification results were normalized using the ribosomal protein S7 gene as an internal control.
7
Real-time PCR Quantification of Gene Expression
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One microgram of total RNA was reverse-transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen). As a control for genomic DNA contamination, the same reaction was performed without Reverse Transcriptase. Real-time PCR was performed in a final volume of 20 μl containing 20 ng of cDNA using SYBR Fast Universal qPCR Kit (Kapa Biosystems) and analyzed using the Quant Studio 6 Flex system (Applied Biosystems). The real-time PCR conditions is one hold at [95 °C, 180 s], followed by 40 cycles of [95 °C, 1 s] and [60 °C, 20 s] steps. After amplification, a melting-curve analysis was done to verify PCR specificity and the absence of primer dimers. For quantification, the abundance of each gene was determined relative to the house-keeping transcript ACT1 and the relative gene expression between the DMSO control and rapamycin-treated conditions were calculated using the 2−ΔΔCt method [35 (link)]. A list of primers used for real-time qRT-PCR is provided in Additional file 10 : Table S7.
8
Quantitative Gene Expression Analysis
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Total RNA was extracted from cultured cell lines using Trizol reagent (Invitrogen) and subjected to reverse transcription using a cDNA Synthesis Kit (Thermo, K1622). Real-time qPCR was performed using a SYBR FAST Universal qPCR Kit (KAPA, KK4602). The relative expression levels of the target genes were calculated as two power values of ΔCt (the Ct of b-actin or PBK minus the Ct of the target gene). The sequences of the PCR primers used for amplification were as follows:
β-actin forward, 5′- AAGGTCATCC CTGAGCT GAA -3′;
β-actin reverse, 5′- TGACAAAGTG GTCGTTG AGG -3′;
PBK forward, 5′- GAAGAGGACTGAGAGTG GCT -3′;
PBK reverse, 5′- CTTCTGCATAAACGGAGA GGC -3′.
β-actin forward, 5′- AAGGTCATCC CTGAGCT GAA -3′;
β-actin reverse, 5′- TGACAAAGTG GTCGTTG AGG -3′;
PBK forward, 5′- GAAGAGGACTGAGAGTG GCT -3′;
PBK reverse, 5′- CTTCTGCATAAACGGAGA GGC -3′.
9
Quantifying Gene Expression via RT-qPCR
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Total RNA was extracted by Rneasy Mini Kit (Qiagen), and cDNA was isolated by GoScript Reverse Transcription System (Promega). Real-time quantitative polymerase chain reactions (RT-qPCR) were set up using the SYBR FAST Universal qPCR kit (KAPA). Relative expression values were normalized to Gapdh expression, and data was processed using the comparative Ct method. Each experiment was performed with technical triplicates. The primers used are listed in Supplementary Table 1 . Significant differences between groups were determined using t-test, and P-values < 0.05 were considered statistically significant.
10
Primer Design and qPCR Validation for AAAP Genes
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The qPCR primer for examined AAAP genes was designed using Primer Premier (v5.0) software (Premier Biosoft, Rockville, MD, USA) (Table S4 ). To ensure the specific amplification of the intended target genes in BPHs, all qPCR primers used in the following study were firstly checked using the online software tool of Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast ) prior to qPCR experiments [47 (link)] (checked on 15 February 2021). The qPCR system of the 10 μL reaction sample consisted of 1 μL cDNA, 0.3 μL forward and reverse primers (concentration of 10 μmol/L), 5 μL SYBR FAST Universal qPCR mix (KAPA Biosystems, Woburn, MA, USA), and 3.4 μL ddH2O. qPCR assays were conducted using a Light Cycler 480 System (Roche Diagnostics, Basel, Switzerland) and the SYBR FAST Universal qPCR Kit (KAPA Biosystems, Woburn, MA, USA), with a PCR amplification procedure as follows: one cycle at 95 °C for 5 min, followed by 45 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. For the entire experiment, three biological replicates and three technical replicates were used for each treatment.
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