Total RNA was isolated from homogenized skin tissue using ISOGEN Reagent (Nippon Gene, Tokyo, Japan) according to the instructions from the manufacturer. Two micrograms of RNA were used for synthesis of cDNA with a cDNA synthesis kit (Applied Biosystems, Foster, CA, USA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed for skin and lymph node tis™sues with Fast SYBR Green Master Mix in a StepOne real time qPCR system (Applied Biosystems) according to the manufacturer's instructions. The quantity of mRNA for each gene was normalized with that of the housekeeping gene ornithine decarboxylase antizyme-1 (Oaz1). The primer sequences and PCR conditions for each of the genes Ifng, Tnf, Il10, Tgfb1, Cd29, Cd90, Cd105 and Oaz1 are described in Table 1 .
Stepone real time qpcr system
Manufactured by Thermo Fisher Scientific
The StepOne real-time qPCR system is a compact, easy-to-use instrument designed for real-time quantitative PCR (qPCR) analysis. It features a high-resolution optical system and supports a range of fluorescent dyes and probe chemistries for accurate gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Isogen reagent, by Nippon Gene (1 mentions)
Universal probe library, by Roche (1 mentions)
Biotek synergy microplate reader, by Agilent Technologies (1 mentions)
Ribopure kit, by Thermo Fisher Scientific (1 mentions)
Rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using stepone real time qpcr system
1
Comprehensive Gene Expression Analysis in Skin and Lymph Node Tissues
2
Gene Expression Profiling of Myogenesis
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Total RNA isolation was conducted using a RiboPure kit (Applied Biosystems, Foster City, State, USA). Total RNA concentration was determined using a BioTek Synergy microplate reader (BioTek Instruments, Winooski, VT, USA). Complementary DNA (cDNA) was constructed by reverse transcribing mRNA, using a RNA-to-cDNA Kit (Applied Biosystems). Transcription of Cyclin D1, Ki-67, C-fos, Myod, Myogenin, Mrf4, α-actin, Il-6, Gapdh, and 18S, were quantified by RT-qPCR, using a StepOne Real-Time qPCR system (Applied Biosystems) and primers from the Universal Probe library (Roche diagnostics, Almere, The Netherlands), or Taqman gene expression assays (for Il-6 and Gapdh; Applied Biosystems). All mRNA expression levels were normalized for 18S and Gapdh expression levels. The primer sequences for each gene are listed in Table 1.
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