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).
7500 fast real time pcr sequence detector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7500 Fast Real-time PCR Sequence Detector is a laboratory instrument designed for the detection and analysis of nucleic acid sequences. It utilizes real-time PCR technology to amplify and quantify target DNA or RNA samples with precision. The core function of this product is to provide accurate and efficient nucleic acid detection and quantification for research and diagnostic applications.
Automatically generated - may contain errors
3 protocols using 7500 fast real time pcr sequence detector
1
Validating Gene Expression via Real-Time PCR
2
Quantitative RT-PCR Analysis of Aortic Gene Expression
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Suprarenal aortas and perivascular and white adipose tissue were homogenized with Tissue Lyser using safe-lock tubes with metal bead and trizol-chloroform. Total aortic RNA was isolated with RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse transcribed with random primers and Superscript III (Invitrogen, Carlsbad, CA, USA). cDNA (1.0 ng) was amplified by RT-PCR with TaqMan Universal PCR Mastermix (Applied Biosystems, Foster City, CA, USA) using 7500 Fast Real-time PCR Sequence Detector (Applied Biosystems)11 (link)51 (link). Samples were run in duplicate and semi-quantified against a standard curve. All probes were obtained from Applied Biosystems and results were normalized to mouse TBP.
3
Aortic Gene Expression Profiling
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Suprarenal aortas were homogenized with Tissue Lyser using safe-lock tubes with metal beads and trizol-chloroform. Total RNA from the aorta was isolated with the RNeasy Mini kit (Qiagen, Hilden, Germany) and reversely transcribed with random primers and Superscript III as described by the manufacturer (Invitrogen, Carlsbad, CA, USA). cDNA (1.0 ng) was amplified by RT-PCR reactions with TaqMan Universal PCR Mastermix (Applied Biosystems, Foster City, CA, USA) in 96-well fast plates on a 7500 Fast Real-time PCR Sequence Detector (Applied Biosystems). Samples were run in duplicate and semi-quantified against a standard curve. All probes were obtained from Applied Biosystems (Supplementary table I) and results were normalized to values of human RPLP0 or murine TBP.
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