Real-time PCRs were performed according to Pirc et al. (40 (link)) in a LightCycler 480 (Roche) using the TaqMan Universal master mix (Applied Biosystems) and the target gene amsC. The final reaction volume (10 μl) contained 0.9 μl of 10 μM primer Ams116F (5′-TCCCACATACTGTGAATCATCCA -3′), 0.9 μl of 10 μM primer Ams189R (5′-GGGTATTTGCGCTAATTTTATTCG -3′), 0.2 μl of 10 μM Ams141T (5′-FAM-CCA GAA TCT GGC CCG CGT ATA CCG-TAMRA-3′), 1 μl ddH2O, 5 μl of 2× TaqMan Universal master mix (Applied Biosystems), and 2 μl of template DNA. All PCRs were conducted in triplicate and negative controls were included. The baseline was set automatically, cycling conditions were: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. A crossing point (Cp) value above 38 would have been considered negative.
Taqman universal master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Spain, France, Canada, Australia, Japan, Belgium
TaqMan Universal Master Mix is a ready-to-use solution designed for qPCR applications. It contains all the necessary components, including DNA polymerase, buffer, and dNTPs, to perform real-time PCR reactions.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (90 mentions)
Trizol reagent, by Thermo Fisher Scientific (85 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (82 mentions)
Rneasy mini kit, by Qiagen (78 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (49 mentions)
Taqman probe, by Thermo Fisher Scientific (45 mentions)
Trizol, by Thermo Fisher Scientific (40 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (38 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (25 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (21 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (21 mentions)
Rneasy kit, by Qiagen (21 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (20 mentions)
Nanodrop, by Thermo Fisher Scientific (20 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (19 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (19 mentions)
Steponeplus, by Thermo Fisher Scientific (18 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (16 mentions)
Iscript cdna synthesis kit, by Bio-Rad (16 mentions)
Rneasy plus mini kit, by Qiagen (15 mentions)
735 protocols using taqman universal master mix
1
Real-Time PCR for amsC Gene Detection
2
Quantitative RT-PCR Assay for NAT1 and NAT2 Genes
3
Quantification of Viral Particles by qPCR
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Infectious virus stocks were titrated on NB-324k cells by plaque assay or by infected cell hybridization assay [27 (link)]. Virus titers are expressed as plaque forming units (PFU) or replication units (RU) per milliliter of virus suspension. Full viral particles were quantified either by dot blot hybridization assays [29 (link)] or by quantitative real-time PCR. Briefly, viral DNA was extracted from iodixanol-purified virus stocks or subcellular fractions using QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. The DNA was eluted in nuclease-free water (Life Technologies, Carlsbad, CA, USA) and stored at −20 °C until measurement. A linearized pH1 plasmid in serial dilutions in nuclease-free water was used to standardize the qPCR. Quantification of viral DNA was carried out by real-time qPCR in a volume of 20 µL using TaqMan® Universal Master Mix (Life Technologies, Carlsbad, CA, USA) supplemented with 0.3 µM of NS-specific primers and a dual-labeled TaqMan® probe (5′-6-FAM and 3′-MGB, Europhins MWG, Ebersberg, Germany). PCR cycles were performed in white 96-well plates (Hard-Shell® PCR plates, Bio-Rad, Hercules, CA, USA) for 40 cycles using a qPCR thermocycler (CFX96 Touch™ Real-Time PCR, Bio-Rad). Virus titers are expressed as the number of viral genomes (Vg) per milliliter of virus suspension.
4
Quantification of miRNA and tRH Levels
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Complementary DNA (cDNA) was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Life Technologies) according to the manufacturer's instructions. Real time PCR amplification was performed using TaqMan Universal Master Mix (Life Technologies) on the Bio-Rad CFX96 real time PCR detection system. U6, miR-24, let-7a, let-7f, RNU48, and RNU66 were all evaluated as potential housekeeping small RNAs for purposes of normalization. RNU48 was selected because it was the most consistent across disease groups. RT-qPCR reactions for human samples were performed in triplicate. RT-qPCR reactions for chimpanzee samples were performed in duplicate. The following TaqMan assays were purchased from Life Technologies: miR-122 (product number 4427975; 002245) and RNU48 or SNORD48 (product number 4427975; 001006). Primers for the custom TaqMan assays (5′ tRH-Gly and 5′ tRH-Val) were designed using 5′-GCAUUGGUGGUUCAGUGGUAGAAUUCUCGCCU-3′ for 5′ tRH-Gly and 5′-GUUUCCGUAGUGUAGUGGUUAUCACGUUCGCCU-3′ for 5′ tRHVal.
5
miR-27b Expression Profiling in Huh7 Cells
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For mRNA analysis, total RNA was extracted from Huh7 cells transfected with miR-27b mimic, miR-27b inhibitor, CM or CI using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to manufacturer instructions. Extracted RNA (1 µg) was reverse-transcribed using iScriptTM Reverse Transcription Supermix (BioRad, Hercules, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed in triplicate using iQ SYBR green Supermix (Bio-Rad) on a Real-Time Detection System (Bio-Rad). The mRNA levels were normalized to ribosomal RNA 18S as a housekeeping gene. Mature miR-27b expression was quantified using TaqMan miRNA Assay kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. qRT-PCR was performed using TaqMan Universal Master Mix (Life Technologies); U6 RNA was used for normalization. Primer information is detailed in Table A1 .
6
Validating miRNA Expression via qPCR
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To confirm the results from miRNA-Seq, we selected some miRNAs of interest to measure using PCR. Total RNA was isolated from AMSCs by the mirVana PARIS total RNA isolation kit (Life Technologies, Carlsbad, CA, USA, Cat# AM1556) according to the kit protocol. RNA concentrations were measured by a NanoDrop Spectrophotometer (NanoDrop, Thermo Fisher Scientific, Inc.). A fixed volume of 5 μL of RNA elute at 1 ng/uL was reverse transcribed by using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Cat# 4366596). For PCR, 1.33 μL of RT product was combined with 10 μL of TaqMan Universal Master Mix (Cat# 4440038), 7.67 μL of H2O and 1 μL of primers, including miR-21, miR-146a, miR-155, and miR-210 (Life Technologies, Cat# 000397, 001097, 002623, and 000512 respectively) to make up a 20 μL reaction. RNU6B (Life Technology Cat# 001093) was included in the assay as reference control. Real-time PCR was carried out on an Applied Biosystems (Foster City, CA, USA) ViiA7 Real-Time PCR system at 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Fold changes of miRNA levels in hypoxia relative to normoxia were calculated using the 2−ΔΔCt method.
7
Metagenomic Profiling of Murine Gut Microbiome
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Metagenomic DNA was extracted from the ileal contents of mice by using the QIAamp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s guidelines and previously published protocols29 (link), 30 (link). Real-time q-PCR was performed using TaqMan® Universal Master Mix (Life technologies) to examine the quality and the quantity of the 16 S rDNA. The variable region 4–6 (V4–V6) of the purified 16 S rDNA gene was amplified with PCR. Sequencing was performed utilizing paired-end Illumina MiSeq sequencing system and reagents according to the manufacturer’s instructions. Raw FASTQ files reflecting forward reads were initially filtered for quality and length (≥200 bp) using QIIME31 . Passing sequences were trimmed of the forward primer, and evaluated for chimeras with UCHIME32 (link). The RDP Classifier software was used to bin 16 S rRNA gene sequences into operational taxonomic units (OTUs), which were defined by clustering at 97% similarity.
8
Quantitative 16S rRNA Gene Analysis
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16S rRNA gene copy number was assessed by quantitative PCR (Q-PCR)
using the 16S rRNA universal primers and TaqMan probes, as previously
described41 .
Briefly, total 16S rRNA gene copy number was calculated against a standard
curve of known 16S rRNA copy numbers
(1×102−1×109). Q-PCR was
performed in triplicate 20 μl reactions containing final
concentrations of 1 ×TaqMan Universal Master Mix (Life Technologies),
100 ng of extracted genomic DNA, 900 nM of each primer, P891F
(5’-seq-3’F) and P1033R (5’-seq-3’R) and 125 nM
of UniProbe under the following conditions: 50 °C for 2 min, 95
°C for 10 min, followed by 40 cycles of denaturation at 95 °C
for 15 s, and annealing and extension at 60 °C for 1 min, along with
no-template control and 8 standards. Copy number was normalized either by
100ng of input DNA, when possible. When too little DNA was obtained, such as
in the case of the buffers, 10μL of DNA extract was added to the PCR
reaction and copy number was normalized by weight of frozen sample.
using the 16S rRNA universal primers and TaqMan probes, as previously
described41 .
Briefly, total 16S rRNA gene copy number was calculated against a standard
curve of known 16S rRNA copy numbers
(1×102−1×109). Q-PCR was
performed in triplicate 20 μl reactions containing final
concentrations of 1 ×TaqMan Universal Master Mix (Life Technologies),
100 ng of extracted genomic DNA, 900 nM of each primer, P891F
(5’-seq-3’F) and P1033R (5’-seq-3’R) and 125 nM
of UniProbe under the following conditions: 50 °C for 2 min, 95
°C for 10 min, followed by 40 cycles of denaturation at 95 °C
for 15 s, and annealing and extension at 60 °C for 1 min, along with
no-template control and 8 standards. Copy number was normalized either by
100ng of input DNA, when possible. When too little DNA was obtained, such as
in the case of the buffers, 10μL of DNA extract was added to the PCR
reaction and copy number was normalized by weight of frozen sample.
9
Quantification of 16S rRNA Gene Copy Number
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16S rRNA gene copy number was assessed by quantitative PCR (Q-PCR) using the 16S rRNA universal primers and TaqMan probes; P891F (5′-TGGAGCATGTGGTT TAATTCGA-3′ ), P1033R (5′-TGCGGGACTTAACCCAACA-3′ ) and UniProbe (5′-FAM-CACGAGCTGACGACARCCATGCA-BHQ-3′; [16] (link)). Total 16S rRNA gene copy number was calculated against a standard curve of known 16SrRNA copy numbers (1×102−1×109). Regression coefficients for all standard curves were >0.99. Q-PCR was performed in triplicate 20 µl reactions containing 1×TaqMan Universal Master Mix (Life Technologies), 20 ng of extracted DNA, each primer at a final concentration of 900 nM and UniProbe at a final concentration of 125 nM under the following conditions: 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. No-template control reactions run in parallel did not produce any detectable signal.
10
Quantitative PCR Analysis of Immune Markers
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To perform PCR, we used 40 ng of cDNA, 0.5 μl of specific primer, 5.0 μl of Taqman Universal Master Mix (4369016, Life Technologies, Carlsbad, CA, USA) and RNase-free water at a final volume of 10 μl. TaqMan gene expression analyses were performed on a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The reference gene was rat GAPDH, cat. number 4352338E (Applied Biosystems, Foster City, CA, USA).
The target gene primers were purchased from Applied Biosystems and included the following: adiponectin (Rn00595250_m1), leptin (Rn00565158_m1), IL-2 (Rn00587673_m1), IL-4 (Rn01456866_m1), IL-6 (Rn01410330_m1), IL-12 (Rn00575112_m1), IFN-α (Rn01400027_g1), IFN-γ( Rn00594078_m1), IGF-1(Rn00710306_m1), and TNF-α (Rn00562055_m1). The control group (MO) average on each treatment day (day 2, 4 or 10) was used to calibrate all groups. PCR efficiency was calculated prior to the experiments, and the 2^-DDCT method was used to calculate gene expression [20 (link)].
The target gene primers were purchased from Applied Biosystems and included the following: adiponectin (Rn00595250_m1), leptin (Rn00565158_m1), IL-2 (Rn00587673_m1), IL-4 (Rn01456866_m1), IL-6 (Rn01410330_m1), IL-12 (Rn00575112_m1), IFN-α (Rn01400027_g1), IFN-γ
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