Taqman universal pcr master mix no amperase

For osteogenesis, the expression levels of Osterix (Sp7) and Runx2 were quantified. For adipogenesis, adiponectin was measured. For chondrogenesis, Sox9 and Col2a were examined. Ribosomal 18S was employed as an internal control/housekeeping gene (all Taqman assayed from Thermo Fisher Scientific).
MicroAmp Optical Reaction Plates (Thermo Fisher Scientific) were employed as the reaction vessel. Master mix was made up for each of the probes/markers that contained 0.5 μL of the specific probe, 5 μL of TaqMan Universal PCR Master Mix No AmpErase (Applied Biosystems), and 3.5 μL of ultrapure water. In each well, a final volume of 9 μL of master mix was mixed with 1 μL of the cDNA. Three replicates were run per sample.
Cycle threshold (CT) value (of the triplicates) for each marker obtained from qPCR data was calculated against the CT values from the 18S housekeeping gene.

For mRNA expression analysis, RNA extraction, cDNA synthesis and qPCR were performed as described previously[11 (link)]. All mRNAs except Chrebpα, Chrebpβ, Srebp-1c were quantified using TaqMan Gene Expression Assays (Applied Biosystems) and TaqMan Universal PCR master mix, No Amp Erase (Applied Biosystems), in a total volume 10 μl. Chrebpα, Chrebpβ, Srebp-1c　mRNA expression levels were quantified using Power SYBR Green PCR master mix (Applied Biosystems) and previously published primers[18 (link)]. Relative mRNA levels of all genes were calculated using the standard-curve method and normalized to the level of cyclophilin A mRNA.
Cyclophilin A gene expression was assessed in all tissues of control and UBC-SKO mice (S2 Fig).

RT-qPCR was employed to quantify the gene expression levels using TaqMan Gene Expression Assays. For osteogenesis, we used the early maker Runx2 (Assay ID Mm00501584-m1) in addition to late osteogenic markers Osteocalcin (OCN) (Assay ID Mm 03413826-mH) and SPARC (Osteonectin) (Assay ID Mm00486332-m1). For Notch target gene expression, we used HES1 (Probe ID Mm01342805-m1) and HEY1 (Assay ID Mm00468865-m1). For early mesodermal differentiation, we used Brachyury (Assay ID Mm00496699-m1). All TaqMan Gene Expression Assays were obtained from Thermo Fisher Scientific. TaqMan Universal PCR MasterMix No AmpErase (Applied Biosystems) was used according to the manufacturer's instructions. StepOnePlus™ Real-Time PCR System was used for running all the samples with the following program: UNG incubation at 50 °C for 2 min; enzyme activation at 95 °C for 20 s; denaturation at 95 °C for 3 s; annealing was performed for 40 cycles at 60 °C for 30 s (40 cycles). The resulting threshold (Ct) values were analyzed with the ΔΔCt method. GAPDH was used as the reference gene, and undifferentiated D3/miPS cells were used as reference samples. 3 biological replicas and 3 technical replicas of each sample were used for the analysis of this test.

Real‐time analyses by two‐step RT–PCR were performed for quantification of miRNA using Thermo Scientific TaqMan chemistry‐based miRNA assay system. It was performed with 25 ng of cellular RNA using specific primers for human let‐7a (assay ID 000377). U6 snRNA (assay ID 001973) was used as an endogenous control. One third of the reverse transcription mix was subjected to PCR amplification with TaqMan^® Universal PCR Master Mix No AmpErase (Thermo Scientific) and the respective TaqMan^® reagents for target miRNA. Samples were analyzed in PCR triplicates from two biological replicates. The comparative C_t method which included normalization, by the U6 snRNA, was used for each cell type for plotting of mean values with SD.