Lumbal DRGs were dissected from mice at indicated time points, and RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Life Technologies). Reverse transcription and real-time PCR were performed using the TaqMan system54 (link) (Life Technologies) and evaluated with the ΔΔC(T) method as described previously55 (link). For G2A and TRPV1, assay primers for the TaqMan system (ThermoFisher) were used.
Taqman system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
The TaqMan system is a real-time PCR technology used for the quantification and detection of nucleic acid sequences. It utilizes fluorescent probe-based chemistry to monitor the amplification of target DNA or RNA during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (22 mentions)
Trizol reagent, by Thermo Fisher Scientific (20 mentions)
Rneasy mini kit, by Qiagen (16 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Icycler iqtm multicolor real time detection system, by Bio-Rad (4 mentions)
Rneasy lipid tissue mini kit, by Qiagen (4 mentions)
Sybr green system, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Abi prism 7300 sequence detection system, by Thermo Fisher Scientific (4 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Rnalater, by Thermo Fisher Scientific (4 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (4 mentions)
Nucleospin rna kit, by Macherey-Nagel (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (3 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (3 mentions)
Step one plus real time pcr thermal cycler, by Thermo Fisher Scientific (3 mentions)
146 protocols using taqman system
1
Quantitative Analysis of DRG Gene Expression
2
RNA Reverse Transcription and qPCR
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Total RNA was reverse-transcribed using a mixture of random primers (High Capacity cDNA Reverse Transcription kit, Applied Biosystems). For brain tissue, 1000 ng of total RNA were reverse-transcribed and the final product was diluted 6 times in RNAse-free water. For RNA of sorted cells, cDNA was pre-amplified (TaqMan® Pre Amp Master Mix (2×) #4384266) with a pool of TaqMan probes, and the final pre-amplified product was diluted 20 times with tris-EDTA (TE) buffer pH 8.0 (#BP2473, Fisher Bioreagents). Quantitative real-time RT-PCR analysis was carried out with Taqman system (#4304437, Life Technology) using the iCycler iQTM Multicolor Real-Time Detection System (Bio-Rad, Hercules, CA, USA).
3
Quantifying TNF-α Gene Expression
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In order to determine TNF-α gene expression (Hs01113624_g1), the normal gastric tissue sample, which was negative for H. pylori infection, was designated as a calibrator. All reactions were repeated, and GUSB (Hs00187320_m1), UBC (Hs00221499_m1), and TBP (Hs00187332_m1) were used as reference genes.
The expression level was measured using the TaqMan® System (Life Technologies, USA) within the Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems™, USA). Relative quantification (RQ) was calculated using the comparative CT method (2-ΔΔct).
The expression level was measured using the TaqMan® System (Life Technologies, USA) within the Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems™, USA). Relative quantification (RQ) was calculated using the comparative CT method (2-ΔΔct).
4
Quantifying Autophagy Markers in Mouse Tissues
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Total RNA was extracted using the RNeasy lipid tissue minikit (74804, Qiagen). Final concentration of 2000 ng/ml was used for reverse transcription into cDNA using reverse transcriptase kit (4374966, LifeTechnologies). cDNA was diluted 1:7 in ultra-pure water (002327777500, BioLab), amplified by the Taqman system (4369016, Life Technologies) and measured by RT-PCR (7500, Applied Biosystems). Relative gene expression was obtained after normalization to endogenous control genes (RPLP0 and HPRT). The following probes were used: Hprt (Mm03024075_m1), RPLP0 (Hs99999902_m1), Atg5 (Mm01187303_m1), Atg12 (Mm00503201_m1), Map1lc3b (Mm00782868_sH), LepR (total isoforms – extracellular domain) (NM_001122899.1) and LepR (long isoform) (NM_146146.2) (ThermoFisher Scientific,Inc).
5
Quantifying HIV Proviral Load by qPCR
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Proviral load was quantified by qPCR using three target sequences synthesized using the TaqMan® system (Life Technologies, Foster City, CA, United States), according to a previously described protocol by Tamegão-Lopes et al. (2006) (link), namely, collection of 5 mL of whole blood for DNA extraction from leukocytes, followed by relative quantification using real-time PCR. The obtained results were further adjusted to an absolute proviral quantification by considering the leukocyte counts per mm3, and the results were expressed as DNA proviral copies/mm3.
6
Investigating Molecular Profiles in Infarcted Brain
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Infarcted brain tissue and a corresponding nonaffected region that, when possible, was the mirror region of the contralateral hemisphere, was dissected out at necropsy, immediately frozen, and kept at −80°C. For RNA extraction, we used TrizolVR Reagent (Life Technologies) followed by PureLinkTM RNA Mini Kit (#12183018 A, Invitrogen), and assessed RNA quantity and quality using a ND‐1000 micro‐spectrophotometer (NanoDrop Technologies). Thousand nanogram of total RNA was reverse‐transcribed and the final product was diluted six times in RNAse‐free water. Real‐time quantitative RT–PCR analysis was carried out with Taqman system (#4304437, Life Technology, Carlsbad, CA, USA) using iCycler iQTM Multicolor Real‐Time Detection System (Bio‐Rad). The Taqman primers used were: PLIN2 (#Hs00605340_m1) and ISG15 (#Hs01921425_s1). GAPDH (#Hs03929097_g1) was used as the housekeeping gene.
7
Quantifying Tendon Gene Expression
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Total RNA was isolated from 75 tendon fragments stored in RNAlater using RNeasy Fibrous Mini Kit (Qiagen) in line with manufacturers' recommended procedures. Reverse transcription reactions were processed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). The relative mRNA expression of VEGF-A, CD34, and FGF-2 was evaluated by real-time PCR using the 7500 Real-Time PCR System, primers, and probes of the TaqMan system (all from Life Technologies, Carlsbad, CA, USA). The primers and probes used in the reactions included Rn01511601_m1 for VEGF-A, Rn03416140_m1 for CD34, Rn00570809_m1 for FGF-2 and Rn99999916_s1 for GAPDH. Real-time PCR reactions were performed on 96-well plates (Life Technologies) at a set time and temperature, including polymerase activation at 50°C for 2 minutes, preliminary denaturation at 94°C for 10 minutes, denaturation at 94°C for 15 s, and annealing of primers, probes, and synthesis at 60°C for 1 min at 40 cycles. The results were standardized based on expression of GAPDH and the relative expression (RQ) of the studied genes was calculated using the ΔΔCt method [19 (link)]. The sample characterized by the lowest ΔCt was used as a calibrator in all the calculations.
8
Quantification of Adipocyte Gene Expression
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Total RNA from epididymal adipocytes or MEFs adipocyte-like cells was extracted using the RNeasy lipid tissue mini-kit (74804, Qiagen) and quantified using nano-drop. Then, 2 μg of RNA were reverse-transcribed with high capacity cDNA reverse transcriptase kit (4374966, Life Technologies Inc.). Taqman system (4369016, Life Technologies, Inc.) was used for real-time PCR amplification. Extraction of total RNA from paired human-AT biopsies (Leipzig cohort) was done according to a previously described protocol [18] (link). Relative gene expression was obtained after normalization to endogenous control genes, using formula 2−ΔΔCt and specific primers (Supplementary Table 1 ).
9
Quantifying Gastric TLR9 mRNA Expression
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The relative expression levels of TLR9 mRNA were measured by real-time quantitative PCR based on the TaqMan® System (Life Technologies, Carlsbad, CA, United States) within the StepOne Plus Real-Time PCR system 2.2.3 (Applied Biosystems) using a specific TaqMan probe for TLR9 (H00002973_m1) and two reference genes, GUSB (Hs00187320_m1) and TBP (Hs00187332_m1) which were tested in a previous study[19 (link)]. A pool of normal H. pylori-negative gastric tissue samples was used as a calibrator. All reactions were performed in triplicate and included a negative reaction control. To determine the amplification efficacy of the probe, a standard curve was constructed with serial dilutions of cDNA samples (pool of pure cDNA and 1:5, 1:25, 1:125, and 1:625 dilutions). Relative quantification (RQ) was calculated using the comparative CT method (2(-∆∆Ct))[20 (link)], while using a pool of the normal mucosa samples as calibrator and reference genes for normalization. The data were expressed as median values.
10
Quantifying TLR2 Expression in Gastric Tissue
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Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. RNA concentration and quality were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States) and a Bioanalyzer (Agilent, United States). A reverse transcription reaction was performed using a High Capacity cDNA kit (Applied Biosystems, Foster City, CA, United States) according to the protocol instructions.
Quantitative PCR was performed with a TaqMan® System (Life Technologies, United States) and the StepOne Plus Real-Time PCR system 2.2.3 (Applied Biosystems, United States) using a TaqMan probe specific for the TLR2 gene (Hs00610101_m1) and two reference genes, GUSB (Hs00187320_m1) and TBP (Hs00187332_m1). The reactions were performed in triplicate and included a negative control. Relative quantification (RQ) was performed using the 2-ΔΔCt method[24 (link)] after normalization to both reference genes, and 14 normal H. pylori-negative gastric tissue samples (C group) were used as a calibrator (RQ = 1.0). RQ was also performed for the samples stratified by polymorphism genotype (at least one polymorphic allele vs wild-type homozygote) and H. pylori infection (negative vs positive). The data were expressed as median values.
Quantitative PCR was performed with a TaqMan® System (Life Technologies, United States) and the StepOne Plus Real-Time PCR system 2.2.3 (Applied Biosystems, United States) using a TaqMan probe specific for the TLR2 gene (Hs00610101_m1) and two reference genes, GUSB (Hs00187320_m1) and TBP (Hs00187332_m1). The reactions were performed in triplicate and included a negative control. Relative quantification (RQ) was performed using the 2-ΔΔCt method[24 (link)] after normalization to both reference genes, and 14 normal H. pylori-negative gastric tissue samples (C group) were used as a calibrator (RQ = 1.0). RQ was also performed for the samples stratified by polymorphism genotype (at least one polymorphic allele vs wild-type homozygote) and H. pylori infection (negative vs positive). The data were expressed as median values.
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