The total RNA was extracted from the hypothalamus, liver, and spleen using Trizol® Reagent (Invitrogen Corporation, CA, USA) according to the manufacturer's recommendations, and quantitated using a Nanodrop ND-2000 (Thermo Electron, WI, USA). Reverse transcription was performed with 3 μg total RNA and a High Capacity cDNA Reverse Transcription kit (Life Technologies Corporation, Carlsbad, CA, USA). Relative expression was determined using the TaqMan™ detection system and primers for the target genes: Mm01312230_m1 for CHRNA7; Mm00443258_m1 for TNF-α; Mm00434228_m1 for IL-1β; Mm00446190_m1 for IL-6; Mm01288386_m1 for IL-10; Mm00657889_mH for Chil3; Mm00436450_m1 for CXCL2; Mm00441242_m1 for CCL2; and Mm00436454_m1 for CX3CL1 (Life Technologies Corporation, Carlsbad, CA, USA). GAPDH was used as the endogenous control (4352339E mouse GAPDH, Life Technologies Corporation, Carlsbad, CA, USA). Each PCR reaction contained 20 ng cDNA. Gene expression was quantitated by real-time PCR performed on an ABI Prism 7500 Fast platform. Data were analyzed using a Sequence Detection System 2.0.5 (Life Technologies Corporation, Carlsbad, CA, USA), and expressed as relative values determined by the comparative threshold cycle (Ct) method (2–ΔΔ Ct), according to the manufacturer's recommendations (16 (link)).
Taqman detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan detection system is a real-time PCR technology developed by Thermo Fisher Scientific. It utilizes fluorescent probes to detect and quantify specific DNA sequences during the amplification process. The system enables sensitive and precise measurement of target molecules in a sample.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sequence detection system 2, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (2 mentions)
Mm00434228 m1, by Thermo Fisher Scientific (2 mentions)
High pure rna isolation kit, by Roche (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Mm00443258 m1, by Thermo Fisher Scientific (1 mentions)
Mm00446190 m1, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
M mulv reverse transcriptase, by New England Biolabs (1 mentions)
M mulv, by New England Biolabs (1 mentions)
Universal pcr master mix reagents kit, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnazol rt, by Molecular Research Center (1 mentions)
Isolate 2 genomic dna kit, by Meridian Bioscience (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
11 protocols using taqman detection system
1
Quantifying Gene Expression in Tissues
2
Validation of Differentially Expressed miRNAs
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In order to validate the expressions of differential miRNAs, quantitative real-time PCR (qRT-PCR) were utilized to detect the relative expression of differentially-expressed miRNAs, with samples enlarged in each group (n = 6 to 8 per group) using the TaqMan detection system (Life Technologies, Foster City, CA, USA). All of the mice in each group were included for the validation. Total RNA was reversely transcribed using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Life Technologies, Foster City, CA, USA). All of the miRNA-specific reverse-transcription primers were provided with the TaqMan MicroRNA Assay and purchased from Life Technologies Corporation (Applied Biosystems, Life Technologies, Foster City, CA, USA). The primers were mmu-miR-615 (ID 2353), mmu-miR-124 (ID 2197), mmu-miR-376b (ID 2452), mmu-let-7e (ID 2407), mmu-miR-708 (ID 1643), and mmu-miR-879 (ID 2473). U6 small nuclear RNA (ID 1973) was used as an endogenous control. Gene expression was quantified by qRT-PCR and performed on an ABI prism Vii7 Sequence Detection System platform (ABI Prism® Vii7, Applied Biosystems, Life Technologies, Foster City, CA, USA). Data were analyzed and the fold change was calculated using the comparative Ct method. All reactions were carried out with three biological replicates, and each analysis consisted of three technical replicates.
3
Extraction and Quantification of Gene Expression
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The total RNA was extracted from the hypothalamus, liver, spleen, isolated bone marrow cells, intraperitoneal macrophages, and cell line BV-2 using Trizol® Reagent (Invitrogen Corporation, CA, USA) according to the manufacturer's recommendations and quantitated using a ND-2000 Nanodrop (Thermo Electron, WI, USA). Reverse transcription was performed with 3 μg of the total RNA, using a High Capacity cDNA Reverse Transcription kit (Life Technologies Corporation, Carlsbad, CA, USA). The relative expression was determined using the TaqMan™ detection system, and the primers for the target genes were obtained from Applied Biosystems: Mm01312230_m1 for CHRNA7; Mm00446190_m1 for IL-6; Mm00434228_m1 for IL-1β; and Mm00443258_m1 for TNFα. GAPDH (4351309) or β-actin (4351315) was used as endogenous controls. Gene expression was quantitated by performing real-time PCR on an ABI Prism 7500 Fast platform. The data were analyzed using a Sequence Detection System 2.0.5 (Life Technologies Corporation, Carlsbad, CA, USA) and expressed as relative values determined by the comparative threshold cycle (Ct) method (2–ΔΔ Ct), according to the manufacturer's recommendations.
4
Quantifying Muscle Lactate Transporter Expression
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Total RNA was extracted from the white gastrocnemius and soleus muscles using Trizol reagent (Life Technologies Corporation, CA, USA) according to the manufacturer's recommendations. Total RNA was quantified using a Nanodrop ND-2000 (Thermo Scientific, WI, USA). Reverse transcription was performed with 3 μg of total RNA using a High-Capacity cDNA Reverse Transcription kit (Life Technologies Corporation, California, USA). The messenger RNA (mRNA) was measured using primers and a TaqMan detection system obtained from Applied Biosystems: MCT-1 (Slc16a1, GenBank accession NM_012716 ) and MCT-4 (Slc16a3, GenBank accession NM_030834 ). Glyceraldehyde-3-phosphate dehydrogenase primer (Applied Biosystems, cat n# 4352338E) was used as an endogenous control. Real-time PCR analysis of the gene expression was performed on an ABI Prism 7500 Fast platform using 20 ng of cDNA. The data were analyzed using a Sequence Detection System 2.0.5 (Life Technologies Corporation, CA, USA) using the comparative threshold cycle method (2−ΔΔCt), according to the manufacturer's recommendation.
5
Quantification of miR-122 and miR-370
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The miR content was extracted and purified from the Hepa1c1c7 and HepG2 cells and from the liver (150 mg) of the mice using the mirVana miRNA isolation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The relative expression of miR-122 and miR-370 (ID 002245 and ID 002275, respectively, Thermo Fisher Scientific) was determined using primers with a TaqMan detection system and U6 spliceosomal RNA (ID 001973, Thermo Fisher Scientific) as endogenous controls. Gene expression was accessed by real-time PCR performed on the ABI Prism 7500 Fast platform and analyzed according to 2.5.
6
Eag1 Gene Expression Analysis
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Total RNA was isolated with TRIzol reagent (Sigma-Aldrich). cDNA was obtained from RNA (5 μg) previously treated with DNAse I and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). Eag1 gene expression was determined by real-time reverse transcription polymerase chain reaction (RT-PCR) using the TaqMan™ detection system (Thermo Fisher Scientific, Waltham, MA, USA). Hypoxanthine-guanine phosphoribosyl transferase was used as the internal standard. Data were analyzed by the 2−ΔΔCt method and by the threshold cycle.
7
Eag1 Expression Analysis by RT-PCR
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Total RNA was extracted from cell cultures with TRI-zol reagent. Five micrograms of total RNA was reverse transcribed using the Moloney Murine Leukemia Virus Reverse transcriptase (M-MuLV) (New England BioLabs Inc., Ipswich, MA, USA). RT-PCR was performed with 1 μL of cDNA using the TaqMan™ detection system (Thermo Fisher Scientific) and the Universal PCR Master Mix reagents kit (Thermo Fisher Scientific). Probes previously developed from TaqMan were used to study Eag1 (ID: Hs00924320_m1) and Gusb (ID: Hs00939627_m1, as a constitutive gene) expression. The PCR reaction protocol was 95°C for 15 seconds and 60°C for 1 minute (40 cycles). Data were analyzed with the 2−ΔΔCt method.
8
Validation of HBV and HCV qPCR Assays
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For validation of the assay, the international panel cited above was used in qPCR reactions to construct standard curves of HBV DNA or HCV RNA in the following concentrations: HBV 7 or HCV 7 (5,000,000 IU/mL), HCV 6 or HBV 6 (500,000 IU/mL), HBV 5 or HCV 5 (50,000 IU/mL), HBV 4 or HCV 4 (5000 IU/mL), HBV 3 or HCV 3 (500 IU/mL), and HBV 2 or HCV 2 (50 IU/mL). The reactions were performed in a 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the TaqMan detection system (Thermo Fisher Scientific, Waltham, MA, USA) with 12.5 μL of TaqMan Universal Master Buffer, predetermined concentrations of the primer–probe sets cited above, and 50 - 100ng of DNA or cDNA, for a total final volume of 25 μL per reaction. All reactions were performed in duplicate using universal conditions: 50 °C for 2 min, 95 °C for 10 min, 45 cycles of 95 °C for 15 s, and 60 °C for 1 min. Results were analyzed using 7500 Fast Software v. 2.1 (Life Technologies, Carlsbad, CA, USA) and expressed in IU/mL. The baseline and threshold values were automatically adjusted for each test.
9
Quantitative Analysis of RNA and microRNA
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Total RNA and microRNA were extracted from liver (~ 150 mg) or cells using RNAzol RT (Molecular Research Center, MRC, Cincinnati, OH—USA) according to the manufacturer’s recommendations, and quantified using NanoDrop ND-2000. Reverse transcription was performed with 3 μg of total RNA or miRNA by specific reverse transcription kits (Thermo Fisher Scientific, Waltham, Massachusetts—USA). The relative expression of mRNAs (Prkaa2 ID Mm01264789_m1, Lin28a ID Mm00524077_m1) and microRNAs (Let-7a ID 000377, U6srRNA ID 001973) was determined using a Taqman detection system (Thermo Fisher Scientific, Waltham, Massachusetts—USA). qPCR was performed on an ABI Prism 7500 Fast platform, and data were expressed as relative values determined by the comparative threshold cycle (Ct) method (2 − ΔΔCt).
10
Quantitative RNA and DNA Analysis in Murine Lung
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