The composition of the microbiota was analyzed by measuring Eubacteria and eight dominant and subdominant bacterial groups, taxa, and species (Bacteroides/Prevotella, Clostridium coccoides , Clostridium leptum , Bifidobacteria, Lactobacillus/Leuconostoc/Pediococcus, Enterobacteriaceae, Escherichia coli , and Faecalibacterium prausnitzii ). Specific regions of the 16S rRNA genes were detected using specific primers and probes by real-time qPCR (Quantstudio 3; Applied Biosystem by Thermo Fisher Scientific, USA) with either TaqMan universal PCR 2× master mix (Applied Biosystems, USA) or with Takyon Rox SYBR MasterMix dTTP blue (Eurogentec, Belgium). The primers and methods are described in reference 33 (link). Each reaction was run in duplicate, and qPCR experiments were performed according to the following method: 1 cycle at 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. For SYBR green amplification, a melting curve was added to show the amplification specificity. Standard curves of reference strains from bacterial genomic DNA were generated by plotting cycle threshold (CT) versus cell number of reference strain in the qPCR assay, as described in reference 33 (link). Results are expressed as number of bacterial cells/milliliter of colonic medium (34 ).
Takyon rox sybr mastermix dttp blue
Manufactured by Eurogentec
Sourced in Germany, Belgium
Takyon Rox SYBR MasterMix dTTP Blue is a qPCR reagent designed for real-time PCR amplification and detection. It contains SYBR Green I dye, Rox passive reference dye, and dTTP for use in quantitative gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (6 mentions)
Applied biosystems quantstudio 5, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (4 mentions)
Dnase treatment, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Primer express 3, by Thermo Fisher Scientific (1 mentions)
Steponeplus pcr system, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Invisorb fragment cleanup kit, by Stratec (1 mentions)
Steponeplus cycler, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Ariamx real time pcr cycler, by Agilent Technologies (1 mentions)
Steponeplus real time pcr machine, by Thermo Fisher Scientific (1 mentions)
17 protocols using takyon rox sybr mastermix dttp blue
1
Quantifying Gut Microbiome Composition
2
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. First strand cDNA synthesis and qRT-PCR was performed using Takyon One-Step kit converter – Euroscript II and Takyon Rox SYBR MasterMix dTTP Blue (Eurogentec). Real-time quantitative PCR was performed (Applied Biosystems 7500) using the cycling conditions: 48 °C for 10 min (reverse transcription); 95 °C for 3 min (Initial denaturation); 40 cycles of 95 °C for 10 secs, 60 °C for 30 secs, 72 °C for 30 secs. All qRT-PCRs were set up in duplicates using three biological replicates for each sample. The PCR primers used for qRT-PCR are included in Supplementary Table T1.
3
Transcriptomic Analysis of iPSC-Derived Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from iPSC-derived microglia (iMG) and/or BV2 using Direct-zol RNA MiniPrep Kit (Zymo research) according to the manufacturer’s protocol. For RT-qPCR analysis, RNA was reverse transcribed into cDNA using iScript cDNA synthesis kit (BIO-RAD) according to the manufacturer’s protocol. Target specific PCR primers for mouse and human (Supplementary Table 3) were obtained from IDT. For qRT-PCR analysis Takyon Rox SYBR MasterMix dTTP blue (Eurogentec) was used. For relative expression analysis, the ΔΔCt comparative method was used to compare TBP-normalised expression level of the target mRNA. Data were normalised either to control (BV2) or WT groups (iMG) and are shown as median ± SD. Statistical significance for each gene was assessed using a Kruskal–Wallis test.
4
Quantification of Mitochondrial DNA from Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total DNA from plasma was isolated using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For quantification of mitochondrial DNA (mtDNA) Real-time quantitative PCR was performed on a StepOne Plus PCR System (Applied Biosystems, Darmstadt, Germany) using the Takyon ROX SYBR MasterMix dTTP Blue (Eurogentec, Seraing, Belgium) and the following cycling conditions: 5 min at 95 °C followed by 40 cycles of 3 s at 95 °C and 40 s at 60 °C. A meltcurve was performed after each run. The primers for ATPase 6 (5’-TCCCCATACTAGTTATTATCGAAACCA-3′ and 5′-GCCTGCAGTAATGTTAGCGGTTA-3′) and D-Loop (5′-TGCACGCGATAGCATTGC-3′ and 5′-AGGCAGGAATCAAAGACAGATACTG-3′) were designed with Primer Express 3.0 Software (Applied Biosystems). For absolute quantification of mitochondrial DNA, serially diluted DNA samples generated from purified specific PCR products (Invisorb Fragment CleanUp Kit, Stratec Molecular, Germany) were used as external standards in each run.
5
Quantitative Real-Time PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from confluent WKPT-0293 Cl.2 cells using High Pure RNA Isolation Kit (Roche) according to the manufacturer’s instructions and 1 μg of total RNA was reverse transcribed into cDNA using First Strand cDNA Synthesis Kit (Thermo Scientific, Schwerte, Germany). Quantitative real-time PCR was carried out using Takyon Rox SYBR MasterMix dTTP Blue (Eurogentec, Cologne, Germany) on a StepOnePlus cycler (Applied Biosystems, Schwerte, Germany). The cycling conditions were 3 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. Primer sequences are given in Table 2 . Gapdh was used as a housekeeping gene. For analysis of relative changes, data was analyzed according to the ΔΔCT method [63 (link)].
6
Quantification of Bacteria, Archaea, and bamA Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The quantification assays were conducted in an AriaMX real-time PCR cycler (Agilent). Total bacteria and archaea abundances were assayed using the protocol described by Yu et al. (2006) . The bamA gene quantification was performed using the primer set Bam-sp9 and Bam-asp1 targeting a 300 bp fragment (Kuntze et al., 2008) . Each 20 u L PCR reaction contained 10 u L of Takyon Rox SYBR MasterMix dTTP Blue (Eurogentec, Köln, Germany), 0.9 u M of each primer and 2 u L of ten fold diluted DNA template. The thermal program consisted of an initial denaturation (95 °C, 3 min) and 44 amplification cycles (95 °C for 3 s; 60 °C for 40 s). Melting curves were constructed from 65 °C to 95 °C, read every 0.5 °C for 5 s. Calibration curves of bamA gene (10°-10 6 gene copies/uL) were prepared using genomic DNA from the anaerobic phenol-degrader Thauera aromatica (DSM 6984) assuming a genome size of 4.6 Mb (Kazy et al., 2010) and one copy number of this gene per genome. Quantification was carried out on triplicate samples. The qPCR efficiencies for all reactions were between 90-100 %. The results were expressed in gene copy number per gram of wet weight of sludge.
7
Mitochondrial DNA Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (1 × 106) were seeded in 10 cm dishes and after 24h incubation DNA extraction was performed using AllPrep DNA/RNA kit (Qiagen GmbH, Hilden, Germany). 30ng DNA for LS and 0.3ng DNA for B16 cells were used and the ratio of mt/gDNA was determined by quantitative PCR, by amplifying hNADH-dehydrogenase and hLPL (lipoprotein lipase) for the LS174T cells and mCytB (cytochrome b)/mACT (actin) for B16 cells and calculating the ratio of their expression, using ddCt method. Oligonucleotides for hNADH, hLPL, mCytB and mACT were kindly provided by dr D. Pisani (IBV, Nice, France). The qPCR was performed with Takyon™ Rox SYBR MasterMix dTTP Blue (Eurogentec, Seraing, Belgium) on StepOnePlus Real Time PCR Machine (Applied Biosystems, Life Technologies, Villebon-sur-Yvette, France).
8
Transcriptomic Analysis of Salmonella Enteritidis
Check if the same lab product or an alternative is used in the 5 most similar protocols
Salmonella Enteritidis ATCC13076 were cultured in LB medium with 1 mg/ml of the extracts. After 24 hours, the bacteria were centrifuged at 12000 g for 10 minutes, and then the RNA was extracted using 600 μl TRIzol™ Reagent (Invitrogen™) as recommended by the manufacturer. The cDNA was synthesized using the enzyme RevertAid H Minus Reverse Transcriptase (Thermo Scientific™). With the cDNA, rt-qPCR analysis was made on the AriaMx Real-time PCR System (Agilent Technologies) thermocycler with the Takyon™ ROX SYBR® MasterMix dTTP Blue (Eurogentec) kit using the primers described inTable 2 . As housekeeping gene, we use 16S rRNA and the relative expression levels were calculated using the 2−ΔΔCt method.
+ Expand
Salmonella Enteritidis ATCC13076 were cultured in LB medium with 1 mg/ml of the extracts. After 24 hours, the bacteria were centrifuged at 12000 g for 10 minutes, and then the RNA was extracted using 600 μl TRIzol™ Reagent (Invitrogen™) as recommended by the manufacturer. The cDNA was synthesized using the enzyme RevertAid H Minus Reverse Transcriptase (Thermo Scientific™). With the cDNA, rt-qPCR analysis was made on the AriaMx Real-time PCR System (Agilent Technologies) thermocycler with the Takyon™ ROX SYBR® MasterMix dTTP Blue (Eurogentec) kit using the primers described in
9
Quantitative RNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using the Nucleospin RNA XS kit (Macherey-Nagel) from a minimum of 100 handpicked islets per mouse. RNA was reverse transcribed to synthesized cDNA using the high capacity cDNA reverse transcription kit (Thermo Fisher) and measurements were performed by qPCR using Takyon ROX SYBR MasterMix dTTP blue (Eurogentec) on a 7900HT Fast System (Applied Biosystems). Resulting levels of fluorescence were submitted to relative quantification by normalization against a housekeeping gene (GAPDH) and expressed as 2 -(DCT) values.
10
Quantitative RT-PCR Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using a RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. RNA concentration and purity were evaluated by spectrophotometry (NanoDrop 2000c, Thermo Fischer Scientific). A maximum of 500 ng of RNA were reverse transcribed with both oligo dT and random primers using a PrimeScript RT Reagent Kit (Perfect Real Time, Takara Bio Inc.) in a 10 µL reaction. Real-time PCR reactions were performed in duplicate using Takyon ROX SYBR MasterMix blue dTTP (Eurogentec) on an Applied Biosystems QuantStudio 5 (Thermo Fischer Scientific). Transcripts were quantified using the following program: 3 min at 95°C followed by 40 cycles of 15 s at 95°C, 20 s at 60°C and 20 s at 72°C. Values for each transcript were normalized to expression levels of RPL13A (60S ribosomal protein L13a), using the 2-ΔΔCt method. Primers used for quantification of transcripts are indicated within Supplementary Table 2.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!