To validate the gene array results, RNA was reverse transcribed with random primers and Superscript III (Invitrogen, Carlsbad, Calif) in accordance with the manufacturer's instructions and analyzed with real-time polymerase chain reaction (PCR). Each PCR used the standard curve method in a fluorescent temperature cycler. cDNA (1.0 ng) was amplified in a 20-μL PCR using the TaqMan Universal PCR Mastermix (Applied Biosystems, Foster City, Calif) in 96-well fast plates on a 7500 fast real-time PCR sequence detector (Applied Biosystems) in duplicate. The following probes (Thermo Fisher Scientific) were used: CD68 (Mm03047343_m1), Mmp12 (Mm00500554_m1), Clec7a (Mm01183349_m1), Gpnmb (Mm01328587_m1), and Spp1 (Mm00436767_m1). The results were normalized to the values of Tbp (Mm00446973_m1).
Mmp12
Manufactured by Thermo Fisher Scientific
Sourced in United States
Mmp12 is a laboratory instrument used for the analysis and detection of specific proteins or enzymes. It functions as a tool for researchers to measure and quantify the presence and activity of the target analyte in various sample types. The core functionality of Mmp12 is to provide accurate and reliable data to support scientific investigations and biological research.
Automatically generated - may contain errors
Lab products found in correlation
7500 fast real time pcr sequence detector, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Collagen 1, by Abcam (1 mentions)
Srebp1, by Santa Cruz Biotechnology (1 mentions)
Keratin 16, by Santa Cruz Biotechnology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Adiponectin, by Abcam (1 mentions)
Anti mouse igg antibody, by Cell Signaling Technology (1 mentions)
Mmp 1, by Santa Cruz Biotechnology (1 mentions)
Nf kb p65, by Abcam (1 mentions)
Mmp 7, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Direct zol rna microprep kit, by Genesee Scientific (1 mentions)
Rsad2, by Thermo Fisher Scientific (1 mentions)
Qscript cdna supermix, by Quantabio (1 mentions)
Timp1, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Iq5 thermocycler, by Bio-Rad (1 mentions)
3 protocols using mmp12
1
Validating Gene Expression via Real-Time PCR
2
Protein Expression Analysis Protocol
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Protein was extracted using cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Protein content in the lysate was determined using the BCA Protein Assay (Pierce, Rockford, IL, USA). Equal amounts of protein were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to a polyvinylidene difluoride membrane. The following antibodies were used: matrix metalloproteinase (MMP)-1, SREBP-1, and Keratin 16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), nuclear factor kappa B (NF-kB) p65, interleukin (IL)-1α, IL-8, IL-6, collagen1, adiponectin, and phosphorylated insulin- like growth factor 1 receptor (Abcam, Cambridge, MA, USA), phospho ERK, Akt, and PI3K (Cell Signaling Technology), MMP-7, and MMP-12 (Thermo, Pittsburgh, PA, USA). Secondary anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG antibody (Cell Signaling Technology) were used to detect primary antibodies. Films of blots were analyzed and quantified using a densitometric program (TINA, Raytest Isotopenmebgerate, Straubenhardt, Germany). All experiments were repeated a minimum of four times.
3
Gene Expression Analysis of Inflammatory Markers
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Samples were isolated for RNA according to the manufacturer's protocol using Direct-zol™ RNA MicroPrep kit (Genesee Scientific). RNA concentration was obtained by nanodrop, and cDNA was made using qscript cDNA supermix following the manufacturer’s instructions (Quantabio). cDNA was synthesized at 1000 ng using BioRAD iQ5 thermocycler. Cycles were: Priming for 5 min at 25 °C, RT: 30 min at 42 °C and RT inactivation for 5 min at 85 °C. cDNA was used to analyze gene expression by real time polymerase chain reaction (RT q-PCR) using PerfeCTa qPCR FastMixII (QUantbio). TaqMan assays used were Il6, Tnfα, Il1b, Gpnmb, Col1a1, Timp1, Mmp12, Rsad2, Ilrn1, Il1bp and 18S eukaryotic endogenous control (Thermo Fisher Scientific). Samples were normalized to 18S.
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