A TaqMan real-time PCR was established for genotyping of DNA samples. A new assay was standardized for the evaluation of this SNPs by real-time PCR in 25 μL reactions using the primers and probes described in Table 1 (Sangon Biotech). The assays used 2 × Premix Ex Taq (Probe qPCR), 50 × ROX Reference Dye II, (Premix Ex Taq™ (Probe qPCR); TAKARA) 30 ng of genomic DNA, the final concentration of primer and probe is 0.4 μM. Both reactions were performed in a real-time PCR system, ABI 7500 Fast (Applied Biosystems), under the following conditions: a pre-amplification step of 60°C for 1 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min, as well as a post-amplification step of 60°C for 1 min.
Premix ex taq
Manufactured by Takara Bio
Sourced in Japan, China, United States
Premix Ex Taq is a ready-to-use PCR master mix solution developed by Takara Bio. It contains all the essential components required for performing polymerase chain reaction (PCR) experiments, including a thermostable DNA polymerase, dNTPs, and buffer. This product is designed to simplify the PCR setup process and improve consistency in PCR results.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (30 mentions)
Primescript rt reagent kit, by Takara Bio (25 mentions)
Trizol, by Thermo Fisher Scientific (19 mentions)
Rneasy mini kit, by Qiagen (14 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (12 mentions)
Revertra ace, by Toyobo (9 mentions)
Oligo dt, by Takara Bio (9 mentions)
Goscript reverse transcription system, by Promega (9 mentions)
Primescript rtase, by Takara Bio (9 mentions)
Sybr green 1, by Thermo Fisher Scientific (7 mentions)
Tri reagent, by Molecular Research Center (7 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (6 mentions)
Sybr premix ex taq, by Takara Bio (6 mentions)
Cfx96 real time system, by Bio-Rad (6 mentions)
Steponeplus system, by Thermo Fisher Scientific (6 mentions)
Miseq platform, by Illumina (5 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (5 mentions)
Qiaamp dna blood mini kit, by Qiagen (4 mentions)
Rox reference dye 2, by Takara Bio (4 mentions)
Isogen, by Nippon Gene (4 mentions)
266 protocols using premix ex taq
1
TaqMan Real-Time PCR Genotyping Assay
2
TaqMan Protocols for Bacterial Detection
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Two TaqMan protocols were used. The Schaad protocol, described by Schaad et al. (1999) and used in experiments 1-6, was as follows: 2 μl of DNA-extract was added to a mix of ROXII (0.5x), Premix Ex Taq (1x) from Takara (Westburg, Leusden, The Netherlands), forward primer Cs 50-2F and reverse primer Cs 133-R (final concentration 0.3 μM each), probe FAM-Cs 50-53T (final concentration 0.1 μM) to a total volume of 25 μl (experiments 1-3) or 20 μl (experiments 4-6). The second protocol (Nytor protocol), described by Vreeburg et al. (2016) and used in experiments 4-6, was as follows: 2 μl of DNA-extract was added to a mix of ROXII (0.5x), Premix Ex Taq (1x) from Takara (Westburg, Leusden, The Netherlands), forward primer Cm-pan1-F2 and reverse primer Cm-pan1-R3 (final concentration 0.3 μM each), probe TP-Cs-pan1-198 (final concentration 0.1 μM) to a total volume of 20 μl. The real-time PCR amplification was performed using a real-time PCR system QuantStudio (Applied Biosystems BV) with the following cycling conditions: initial denaturation for 2 min at 95 °C; then 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. For analysis of plant material, Ct values lower than 35 were considered positive.
3
Quantifying Mouse Arv1 Gene Expression
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Total RNA was isolated from liver using TriZol (Invitrogen, Grand Island, NY, USA). The QIAGEN RNeasy mini kit was used to isolate RNA from nonhepatic tissues, with the exception of adipose and brain, in which cases the QIAGEN RNeasy lipid tissue mini kit proved superior. RNA (1 μg) was reverse transcribed into cDNA using the SuperScript III Kit (Invitrogen) or the iScript kit (Bio-Rad, Hercules, CA, USA). The cDNA products were diluted 1:20 into a final reaction volume of 12.5 μl in the wells of a 384-well reaction plate containing 300 nm of each forward and reverse primer, and 6.25 μl of 2X SYBR green master mix (Applied Biosystems, Grand Island, CA, USA). The following primers were used to amplify exons 2 and 3 of mouse ARV1: forward 5′-TCAGGAGCTGTACCGGGACTA-3′, reverse 5′-CTGCAGCTGCCACCACCGCAGGTATGCTTCA-3′. Beta actin was amplified with the following primers: forward 5′-TTGGGTATGGAATCCTGTGG-3′, reverse 5′-CTTCTGCATCCTGTCAGCAA-3′. A Taqman assay to exons 2 and 3 was also used to measure Arv1 expression (Mm01253489_m1, Applied Biosystems) with Premix Ex Taq (RR390L, Clontech, CA, USA). Relative quantities were calculated using the delta delta Ct method.13 (link)
4
Quantitative Real-Time PCR for ICOCAV17 Detection
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Blood samples were collected in PAXgene Blood DNA tubes and processed using the QIAamp DNA Blood Mini Kit (Qiagen; Madrid, Spain) following the manufacturer’s instructions. Tumors from necropsies were snap frozen at −80°C as dried pieces. Tissue samples were introduced into a metal cylinder submerged in liquid nitrogen and ground thoroughly with a pestle until they were completely pulverized. DNA was isolated using the QIAamp DNA Mini Qit (Qiagen). DNA quantification and purity (A260/280 and A260/230) were analyzed with a Nanodrop 2000 spectrophotometer (Thermo Scientific). Dilutions of ICOCAV17 from 108 to 103 vp/mL in blood from healthy donors were used for the standard curve. Samples were analyzed in triplicate by the quantitative real-time PCR (qRT-PCR) 7500 Fast (Applied Biosystems; Madrid, Spain) using the Premix Ex Taq (Clontech; Saint-Germain-en-Laye, France), with forward primer (0.5 µmol/L) 5’TGTGGGCCTGTGTGATTCCT-3’; reverse primer (0.5 µmol/L) 5’-CCAGAATCAGCCTGAGTGCTC-3’; and 10 pmol of Taqman probe FAM-CTCGAATCAGTGTCAGGCTCCGCA-TAMRA, which identified the E1A region. The qRT-PCR conditions were as follows: holding stage 10 min at 95°C, cycling stage 15 s at 95°C and 1 min at 60°C, repeated 40 times. Analysis was performed using the 7500 Software V.2.0.6 (Applied Biosystems).
5
Quantifying Circadian Gene Expression
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With β-actin (GenBank accession no.: X97614 ) as the reference gene, Real-time PCR (qPCR) reactions were carried out with the first strand cDNAs using the TaqMan method in 20 μl reaction agent comprised of one μl of template cDNA, 2*Premix Ex Taq™ (Takara, Kusatsu, Japan), 0.2 μM each primer and 0.4 μM probe (Table S1 ) on a 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Thermal cycling conditions were: 45 cycles of 95 °C for 15 s, 60 °C for 34 s. cDNA sample of each group was replicated three times. At least three groups of individuals were tested for each data-point. Fold differences of Hacry1 or Hacry2 transcripts were calculated according to the 2–ΔΔCT method (Livak & Schmittgen, 2001 (link)).
6
Papilloma Skin Lesion DNA Extraction and PCR Analysis
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Briefly, 100 mg of each collected papilloma skin lesion sample were removed using a sterile knife and placed in a tissue grinder. After adding 1 mL of physiological saline, the sample was ground. The homogenate was centrifuged at 3,000 r/min for 1 min, and then 200 μL of supernatant was collected. Total DNA was isolated from the supernatant using a MiniBEST Viral DNA Extraction Kit (TaKaRa Bio, Japan) according to the manufacturer's instructions. The extracted DNA was used as a PCR template. The PCR was carried out using a Bio-Rad C1000 Touch 850W Thermal Cycler (Bio-Rad, USA). The PCR reaction mix included 21 μL of water, 1 μL (0.2 μmol/L) of each FAP59-FAP64 primer, 25 μL of 2× Premix Ex Taq (TaKaRa Bio), and 2 μL of DNA template. The PCR sequence conditions were as follows: 95°C for 5 min followed by 30 cycles of 40 sec at 94°C, 40 sec at 64°C, and 1 min at 72°C, and a final 10 min at 72°C. The PCR products were separated on 2% agarose gel by electrophoresis, and the gels were stained with ethidium bromide (Takara Bio). Gels were then visualized under UV light and photographed. The DNA bands were recovered using an agarose gel DNA fragment recovery kit (Promega, USA) according to the manufacturer's instructions. The purified DNA fragment was then cloned into pMD19-T (Takara Bio) for sequencing.
7
Quantitative Real-Time PCR Analysis of IL-23 Gene
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DNA extracted from biopsy samples was used for quantitative real-time PCR (RT-PCR) on interleukin 23 (IL-23) gene. DNA was amplified with a Stratagene Mx3000P Real-Time PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). Following Taqman gene expression assays were used: Hs00372324_m1 (IL-23A) and Hs999999 m1 (GAPDH, human glyceraldehyde-3-phosphate dehydrogenase). Human GAPDH was used as the housekeeping gene. PCR amplifications were carried out in a 20 μl total volume: 10 μl of 2 × Premix Ex Taq (Takara, Japan), 1 μl of 20 × TaqMan gene expression assay, 0.4 μl of RoX Reference Dye II (Takara, Japan), 4.6 μl of water, and 4 μl of DNA at following PCR conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. PCR reactions were performed in duplicate. The relative abundance of the expression of each gene was calculated by comparing delta cycle thresholds.
8
Microbial Community Profiling via 16S and ITS Amplicon Sequencing
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For archaea and bacteria, the V4 region of the 16S rRNA gene was amplified using the primer pair 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′) (57 (link)). For fungi, the ITS1 region of the fungal internal transcribed spacer (ITS) was amplified with the primer pair ITS1f (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) (57 (link)). PCR amplifications were conducted in a 25-μl system containing 12.5 μl 2× premix Ex Taq (TaKaRa), 0.5 μl of each primer (10 μM), 1 μl (20 ng μl−1) template DNA, and 10.5 μl sterile double-distilled water. The thermal cycling profile for PCR was as follows: an initial denaturation at 95°C for 10 min; 28 cycles of 30 s at 95°C and 1 min at 50°C (for bacteria) or 60°C (for fungi) for annealing; 1 min at 72°C for extension; with a final extension for 10 min at 72°C. The PCR products were checked by electrophoresis on a 1.5% agarose gel and were then purified with AMPure XP beads (Beckman Coulter Inc., Brea, CA, USA) following the manufacturer’s protocol. A subsequent eight-cycle PCR was performed to add the Illumina sequencing adapters and dual-index barcodes for each amplicon. The PCR products were purified with AMPure beads (Beckman Coulter Inc., Brea, CA, USA) and sequenced on an Illumina MiSeq platform.
9
Quantitative Real-Time PCR Analysis of Inflammatory Markers
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Total RNAs from Raw264.7 cells were extracted using ISOGEN (Nippon Gene, Tokyo, Japan) following the manufacturer's protocol. RNAs were reverse transcribed to cDNAs using the PrimeScript TR reagent kit (Takara, Japan), using the following primers: TNF-α: Forward: 5—TGT CTA CTG AAC TTC GGG GTG AT—3, Reverse: 5—AAC TGA TGA GAG GGA GGC CAT—3; INF-γ: Forward: 5—CAA GGC GAA AAA GGA TGC A—3, Reverse: 5—CGG ATG AGC TCA TTG AAT GCT—3; IL-10: Forward: GGG TGA GAA GCT GAA GAC CCT, Reverse: TCA CCT GCT CCA CTG CCT T; HO-1: Forward: CAG GGT GAC AGA AGA GGC TAA GAC, Reverse: TTG TGT TCC TCT GTC AGC ATC AC; TLR-4: Forward r: GAG CTT CAA CCC CTT GAA GAT CT, Reverse: CCA TGC CAT GCC TTG TCT T; OPN: Forward: CCC GGT GAA AGT GAC TGA TTC T, Reverse: GAT TCT GCT TCT GAG ATG GGT CA. Real time PCR was performed in a 25 μl reaction system containing 12.5 μl 2× Premix Ex Taq™ (Takara, Shiga, Japan), 5 μl of primer, 6.5 μl of cDNA. Amplification was conducted on the Applied Biosystem PRISM7700 (ABI Japan, Co., Ltd., Tokyo, Japan). Quantification was determined by the standard curve and 2-ΔΔCt methods.
10
Trypanosome Detection via Fecal PCR
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Five microliters of fecal DNA was used for PCR amplification in a 50 μl reaction with an AccuPrime™ Taq DNA Polymerase System (Cat. No. 12339016, Invitrogen, Carlsbad, CA, USA), as follows: 94 °C for 2 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 68 °C for 1 min. Primers were designed as described previously, targeting the 24S alpha subunit rRNA gene of trypanosomatids and a nested-PCR was subsequently conducted to amplify the T. cruzi-specific region of the same gene using primers D71 and D72 [19 (link), 20 (link)]. PCR products were examined with 2% agarose gel electrophoresis. Additionally, the qPCR reactions were used to detect T. cruzi satellite DNA gene fragments with 2 × Premix Ex Taq™ (Code No. RR390A, Takara Bio Inc., Japan) as described previously [21 (link)]. Primers and probes were synthesized by Sangon Biotech (Shanghai, China).
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