The PRV load of piglets was monitored by the real-time fluorescence quantification PCR (FQ-PCR) of nasal swab and brain tissue. The total DNA was isolated from the nasal swabs and brains tissue by TIANamp Swab DNA Kit (Tiangen Biotech, Beijing, China) and Genomic DNA Extraction Kit (TaKaRa, Tokyo, Japan), respectively. The upstream and downstream primers were 5′- ACAAGTTCAAGGCCC ACATCTAC -3′ and 5′- GTCYGTGAAGCGGTTCGTG AT -3′, respectively, which were used to amplify a 95-bp fragment of the glycoprotein B gene of PRV (GenBank accession no. KJ526438). A 17-bp probe (5′- ACGTCATCGTCACGACC -3′) complementary to an internal region between two primers was selected and labelled with carboxyfluorescein at the 5′ end and with carboxytetramethylrhodamine at the -3′ end. The FQ-PCR was analyzed by using SsoAdvancedTM Universal Probes Supermix (BIO-RAD, Hercules, CA, USA) with a Bio-Rad CFX96TM Manager software system according the method described in our previous study [21 (link)].
Tianamp swab dna kit
Manufactured by Tiangen Biotech
Sourced in China
The TIANamp Swab DNA Kit is a DNA extraction kit designed for the isolation of genomic DNA from various types of swab samples, such as buccal, nasopharyngeal, or oropharyngeal swabs. The kit utilizes a simple and efficient column-based purification method to obtain high-quality DNA suitable for a wide range of downstream applications.
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Lab products found in correlation
Geneamp pcr system 2400, by Thermo Fisher Scientific (2 mentions)
Transstart fastpfu dna polymerase, by Transgene (2 mentions)
Ssoadvanced universal probes supermix, by Bio-Rad (1 mentions)
Genomic dna extraction kit, by Takara Bio (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Du red, by Biosharp (1 mentions)
Abi 3730xl dna analyser, by Thermo Fisher Scientific (1 mentions)
Ptc 200 pcr instrument, by Bio-Rad (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Roche (1 mentions)
Kapa hyperplus kit, by Roche (1 mentions)
Agilent dna 1000 kit, by Agilent Technologies (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Qubit instrument, by Thermo Fisher Scientific (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Quantifluor st, by Promega (1 mentions)
E gel, by Thermo Fisher Scientific (1 mentions)
Fastprep 24 machine, by MP Biomedicals (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
14 protocols using tianamp swab dna kit
1
Quantitative PCR Assay for Monitoring PRV Load
2
DNA Extraction and Genotyping of Blood Samples
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Blood samples were collected from all participants and stored at −80 °C before DNA extraction. Genomic DNA was extracted from venous blood leukocytes using a genomic DNA extraction and purification kit (TIANamp Swab DNA Kit; Tiangen Biotech, Beijing, China). Study participants were genotyped for the SNPs using Sequenom MassARRAY technology (Bioyong Technologies, Beijing, China). All DNA samples passing initial quality checks were plated at a concentration of ≥5 ng/μl for processing on the platform. Quality measures taken into account for genotyped SNPs to be excluded from the subsequent analysis were minor allele frequency (MAF) <0.05, genotyping success <80%, and failed Hardy–Weinberg equilibrium (HWE) test in control samples (p<0.001). Quality control criteria for each SNP were implemented in our data set.
3
Genetic Analysis of TLR4 Polymorphisms
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Buccal swab samples were collected and stored at −20 °C immediately after extraction, then used for DNA extraction with TIANamp swab DNA Kit (TIANGEN BIOTECHNOLOGY, BEIJING, CO., Ltd.), following the instruction within the kit package. Eight SNPs of TLR4 gene (rs11536889 , rs1927906 , rs1927911 , rs2149356 , rs4986790 , rs4986791 , rs2737190 , rs787384 ) were genotyped by the Sequenom Mass ARRAY system (Shanghai Benegene Biotechnology Co. Ltd.), which was based on the MALDI-TOF technology.
4
Genomic DNA Extraction from Swab Samples
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TIANamp Swab DNA Kit (Tiangen Biotech, Beijing) was used to extract genomic DNA following the manufacturer's instruction. The DNA samples were stored at -20°C until use.
5
Genomic DNA Extraction and PCR Amplification
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Genomic DNA was extracted from exfoliated buccal cells using the TIA Namp Swab DNA Kit (TIANGEN Biotech, Beijing, China) according to standard procedures. Polymerase chain reactions (PCRs) were performed in a 25-μl reaction volume containing 1 μl DNA, 12.5 μl GoTaq Green Master Mix (Promega Company, Madison, WI, USA), 1 μl each of two primers (200 ng/l), and 9.5 μl ddH2O. The amplification protocol included cycling at 94°C for 3 min, followed by 35 cycles at 95°C for 30 s, 58°C for 30 s and 72°C for 45 s in a Gene Amp 2400 PCR system (Applied Biosystems, Carlsbad, CA, USA). PCR products were separated by electrophoresis on a 1.8% agarose gel and stained with Du Red (Biosharp, China), then visualized under ultraviolet (UV) trans-illumination. Sizes were determined by comparison with a 50-bp DNA sequencing ladder.
6
Genetic Influence on Cytokine Profiles
7
RNA-seq and Metagenomic Library Preparation
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Total RNA was extracted by TRIzol LS Reagent (ambion®, Thermo Fisher Scientific, USA). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was applied using cDNA Synthesis Kit (Transcriptor First Strand, Roche, Inc., German), and afterward, we constructed a library by Universal V6 RNA-seq library Prep Kit (VAHTS®, Vazyme, China) for sequencing.
We extracted total DNA in tested specimens using a TIANamp Swab DNA kit (TIANGEN®, TIANGEN, China) and constructed a metagenomic library using KAPA HyperPlus Kit (KAPA, Roche, Inc., German) with dual-indexed Adapters. The DNA was fragmented to approximately 250 bp by the enzyme at 37°C for 20 min. After end repair and A-tailing, adapter ligation, post-ligation Cleanup, library amplification, and post-amplification Cleanup, the library was constructed. Agilent DNA 1000 kit (Agilent, Agilent Technology, Inc., USA) was used for library quality control.
We extracted total DNA in tested specimens using a TIANamp Swab DNA kit (TIANGEN®, TIANGEN, China) and constructed a metagenomic library using KAPA HyperPlus Kit (KAPA, Roche, Inc., German) with dual-indexed Adapters. The DNA was fragmented to approximately 250 bp by the enzyme at 37°C for 20 min. After end repair and A-tailing, adapter ligation, post-ligation Cleanup, library amplification, and post-amplification Cleanup, the library was constructed. Agilent DNA 1000 kit (Agilent, Agilent Technology, Inc., USA) was used for library quality control.
8
16S rRNA Gene Amplicon Sequencing
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Genomic DNA was extracted using a TIANamp Swab DNA kit (Tiangen Biotech, China). The V3-4 hypervariable region of the 16S rRNA genes was amplified using the primers 338F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′) with a TransStart Fastpfu DNA Polymerase (TransGen, Beijing, China). Cycling conditions were as follows: 5 min at 95°C; 20 cycles of 45 s at 95°C, 30 s at 55°C, and 30 s at 72°C; and a final extension step for 10 min at 72°C. Each sample was PCR amplified in triplicate. All amplicons were purified with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and quantified on a Qubit instrument (Life Technologies). Samples were then pooled with equal concentrations, and 2 × 300 bp paired-end sequencing was performed for pooled amplicons on an Illumina MiSeq instrument.
9
Microbial DNA Extraction and 16S Sequencing
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Total genomic DNA was extracted using the TIANamp Swab DNA Kit (TIANGEN, Beijing, China) after TS samples were vortexed with glass beads and TT/NPT samples were liquid nitrogen-ground. To eliminate any contamination caused by blood stains on the surface of the sterile tissues, surface decontamination of the samples was achieved by washing with PBS. Amplification of the 16S rRNA gene V3–4 region was performed with primers 338F and 806R (Huse et al., 2007 ) using TransStart Fastpfu DNA Polymerase (TransGen, Beijing, China). Cycling conditions were as follows: denaturation at 95°C for 2 min; 20 cycles of 45 s at 95°C, 30 s at 55°C, and 30 s at 72°C; and extension at 72°C for 5 min. PCR amplification was performed for each sample in triplicate, and the samples were purified using the AxyPrep DNA Gel Extraction kit (Axygen Scientific Inc., Union City, CA, United States) and assessed via spectrophotometry (QuantiFluor-ST, Promega). Equivalent pooled 16S rRNA PCR amplicons were sequenced on an Illumina MiSeq instrument with 2 × 300 bp paired-end sequencing.
10
Vaginal Bacterial DNA Extraction Protocol
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The TIANamp Swab DNA kit (TIANGEN BIOTECH Beijing Co. Ltd) was used to extract the whole genomic DNA of vaginal bacteria species, according to the manufacturer's instructions. The swab suspensions were mixed with 20 μL proteinase K. The mixture was incubated at 56 °C for 60 min. Then, the samples were subjected to mechanical lysis by bead beating in a Fast Prep 24 machine (MPBio, USA) at 6 m/s for 40 s. The lysates were subjected to centrifugation at 12,000 rpm for 60 s to pellet the beads and filtered using the CR2 columns. DNA was purified, and the quality of total genomic DNA was evaluated by agarose gel electrophoresis (1% E-gel, Invitrogen, USA).
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