Taqman universal pcr master mix

The real-time RT-PCR method with an Assay-on-Demand Gene Expression Product (Life Technologies, P/N 4331182) consisted of unlabeled PCR primers and a TaqMan MGB probe (FAM dye-labeled) optimized to work with the TaqMan Universal PCR Master Mix (P/N 4304437) in an ABI Prism 7700 system (PerkinElmer Life Sciences, Boston, MA, USA) and was employed to quantitatively measure transforming growth factor alpha (TGFA; Hs00608187_m1), transforming growth factor beta 1 (TGFB1; Hs00998133_m1), progesterone receptor (PGR; Hs01556702_m1), tumor necrosis factor receptor superfamily member 9 (TNFRSF9; Hs00155512_m1), bone morphogenetic protein 6 (BMP6; Hs01099594_m1), thyroid hormone receptors α/β (THRA; Hs00268470_m1, and THRB; Hs00230861_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs02758991_g1) mRNA expression (Applied Biosystems). All assays were performed in triplicate. The mRNA contents were normalized to GAPDH mRNA levels, and differences in expression were determined by the CT method described in the ABI user's manual (Life Technologies).

Front legs were stripped of muscle and bones were snap frozen in liquid nitrogen before storage at −80 °C. RNA was extracted from snap frozen front legs using TRIZOL according to the manufacturers protocol. cDNA synthesis was carried out using SuperScript III First Strand Synthesis SuperMix and qPCR was performed using TaqMan Universal PCR Master Mix and an ABI 7900 PCR System (Perkin Elmer, Foster City, CA, USA). Mouse collagen 1α1 and CXCL12 gene expression levels were analyzed using the following TaqMan probes: Col1α1 (Mm00801666_g1) and CXCL12 (Mm00445552_m1). All expression values were calculated using the 2^−∆∆CT method and normalized to GapdH (all reagents from Applied Biosystems, Warrington, UK).

Assay-on-Demand Gene Expression Product (Applied Biosystems, Foster City, CA, USA, 4331182), consisting of unlabeled PCR primers and a TaqMan MGB probe (FAM dye-labeled) optimized to work with the TaqMan Universal PCR Master Mix (P/N 4304437) in an ABI Prism 7700 system (Perkin Elmer Life Sciences, Boston, MA, USA), was employed to quantitatively measure AREG, FBLN1, CLDN6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. All assays were performed in triplicate. mRNA content was normalized to the GAPDH mRNA level and differences in expression were determined by the Ct method described in the ABI user's manual (Applied Biosystems).