Total RNA was extracted from lung tissues and fibroblasts using TRIzol lysis reagent (Life Technologies) and RNeasy Kit (QIAGEN). Reverse transcription was performed with SuperScript IV (Invitrogen). Gene mRNA expression levels were measured by quantitative PCR using the TaqMan real-time PCR system (Life Technologies) according to the manufacturer’s protocol on a TaqMan Gene Expression Assays Step One Plus real-time PCR system (Life Technologies). Each gene’s expression was measured as a ratio to the gene expression of GAPDH or 18S ribosomal RNA (18S). Specific primers and probes for amplifying genes encoding human PLAU (Hs01547054_m1), human SERPINE1 (Hs01126607_g1), human MMP-1 (Hs00899658_m1), human MMP-3 (Hs00968305_m1), human Col1A1 (Hs00164004_m1), human FN (Hs00365052_m1), human ENO (Hs00361415_m1), human ACTA2 (Hs00426835_g1), mouse Fn (Mm01256744_m1), mouse Col1a1 (Mm00801666_g1), mouse Plau, (Mm01274460_g1), mouse Hprt1 (Mm03024075_m1), and mouse Gapdh (Mm99999915_g1) were purchased from Life Technologies.
Mm00801666 g1
Manufactured by Thermo Fisher Scientific
Mm00801666_g1 is a TaqMan Gene Expression Assay designed for quantitative real-time PCR (qRT-PCR) analysis of gene expression in mouse samples. The assay targets a specific gene sequence and provides a standardized and reliable tool for measuring gene expression levels.
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Lab products found in correlation
High capacity cdna archive kit, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression assays mm00725412 s1, by Thermo Fisher Scientific (2 mentions)
Option monitor 3, by Bio-Rad (2 mentions)
Mm03302249 g1, by Thermo Fisher Scientific (2 mentions)
Mm00443258 m1, by Thermo Fisher Scientific (2 mentions)
Trizol rna isolation reagent, by Thermo Fisher Scientific (2 mentions)
Opticon 2 system, by Bio-Rad (2 mentions)
Mm01178820 m1, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Gapdh mm99999915 g1, by Thermo Fisher Scientific (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Trizol lysis reagent, by Thermo Fisher Scientific (1 mentions)
Taqman real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assays step one plus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Hs00426835 g1, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Il 6 mm00446190 m1, by Thermo Fisher Scientific (1 mentions)
Mm99999915 g1, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using mm00801666 g1
1
Transcriptomic Analysis of Lung Fibroblasts
2
Quantifying Gene Expression in Ischemic Kidney
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Total mRNA is extracted from a pole section of the ischemic kidney (PureLink RNA Mini Kit; Life Technologies, Merelbeke, Belgium) and converted to cDNA (High Capacity cDNA archive kit; Life Technologies). To quantify gene expression, qPCR, based on the Taqman fluorescence method (ABI Prism 7000 sequence detection system; Life Technologies), was used. Taqman probes and primers for gapdh (Mm99999915_g1), collagen I 1 (Mm00801666_g1), tgf1 (Mm01178820_m1), dnmt1 (Mm00599763_m1), dnmt3a (Mm00432881_m1) and dnmt3b (Mm01240113_m1), havcr1 (Mm00506686_m1), lcn2 (Mm01324470_m1), tnfα (Mm00443258_m1) and il-6 (Mm00446190_m1) were purchased from Life Technologies. Each gene was analyzed in triplicate and the expression was normalized to the reference gene gapdh. Calculations were made conform the comparative Cq-method.
3
Fracture Callus RNA and Protein Expression
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Total RNA or protein was extracted from fracture calluses after dissection. The proximal and distal borders of the fracture callus are roughly outlined by dashed lines in the low-magnification histological images. For real-time PCR, cDNA template was generated using random hexamers and data were related to the transcript of ribosomal protein 18S as a housekeeping control. Primers for Alp (Mm00475834_m1), BSP (Mm00492555_m1) and Col1 (Mm00801666_g1) were purchased from Applied Biosystems. For western blot analysis, indicated antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL). Relative band intensities were quantified using the ImageJ software ( http://imagej.nih.gov/ij/ ). Anti-β-catenin antibody (working concentration of 1.0 μg ml−l—06-734) was purchased from Millipore, anti-active β-catenin antibody (working concentration of 1.5 μg ml−1—05-665) was purchased from Millipore, anti-β-actin antibody (working concentration of 0.5 ng ml−1—CP01) was purchased from Calbiochem and anti-His antibody (working concentration of 0.2 ng ml−1—s-804) was purchased from Santa Cruz. Full western blot images are shown in Supplementary Fig. 12 .
4
Quantitative Analysis of Hepatic Gene Expression
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TRIzol RNA Isolation Reagent (Life Technologies, Gaithersburg, MD) was used for total RNA extraction from mouse liver [23 (link)]. Total RNA quantity was determined by an absorption spectrometer. The High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) was used for reverse transcription. Real-time RT-PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions, and αSMA, collagen I, IL-1β, IL-6, IL-10, TNF-α, TGF-β and GAPDH were quantified using commercially available kits (TaqMan Gene Expression Assays Mm00725412_s1, Mm00801666_g1, Mm00434228_m1, Mm00446190_m1, Mm00439614_m1, Mm00443258_m1, Mm01178820_m1 and Mm03302249_g1, respectively; Applied Biosystems). These primer sets were designed to span one intron to allow identification of genomic contamination. Target gene results were calculated by delta Ct method (comparing target RNA expression to GAPDH). The reaction protocol consisted of the following cycles: 95°C for 15 min, 95°C for 15 sec and 60°C for 1 min for 50 cycles of PCR amplification on an Opticon 2 System (BioRad). All data were analyzed on an Option monitor 3 (BioRad).
5
Lung RNA Extraction and Expression Analysis
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TRIzol RNA Isolation Reagent (Life Technologies, Gaithersburg, MD, USA) was used for total RNA extraction from the lungs of mice. Total RNA quantity was determined using an absorption spectrometer (NanoDrop 2000 C, Thermo Fisher Scientific, Tokyo, Japan). The High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) was used for reverse transcription. Real-time reverse transcription-PCR was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) according to the instructions provided by the manufacturer. The levels of αSMA, collagen I, TGF-β, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase were quantified using commercially available kits (TaqMan Gene Expression Assays Mm00725412_s1, Mm00801666_g1, Mm01178820_m1, Mm00443258_m1 and Mm03302249_g1 respectively; Applied Biosystems). These primer sets were designed to span one intron to allow the identification of genomic contamination. The reaction protocol consisted of the following cycles: 95 °C for 15 min, 95 °C for 15 s, and 60 °C for 1 min for 50 cycles of PCR amplification on an Opticon 2 System (Bio-Rad). All data were analyzed on an Option monitor 3 (Bio-Rad).
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