Taqman universal pcr master mix no amperase ung

1 X 10⁷ fibroblasts were harvested and total RNA isolated using the Qiagen AllPrep DNA/RNA/Protein mini kit following the manufacturer’s protocol. RNA was quantified by Nanodrop quantitation (Thermofisher Scientific). Complementary DNA (cDNA) was generated from 0.5 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher Scientific) following the manufacturer’s protocol. 10 μL Real Time PCR reactions containing 5 μL 2X TaqMan Universal PCR master mix no AmpErase UNG (Applied Biosystems), 0.5 μL 20X TaqMan Primer–Probe set (ACTB cat #: Hs1060665_g1, POLG cat #: Hs00160298_m1, POLG2 cat #: Hs00945167_m1, Applied Biosystems), and 0.5 μL 1:2 diluted cDNA was setup in a 384-well plate. Real time PCR amplification was performed using a QuantStudio 7 Real-Time PCR instrument (Applied Biosystems) with the cycling parameters at 95°C for 10 min, 95°C 15 sec, 60°C 1 min. The last two steps were performed for 40 cycles. To calculate expression levels and fold change, the following equations were used: ΔCt = Ct_GI-Ct_Actin, ΔΔCt = ΔCt_P- ΔCt_C, FC = 2^-ΔΔCt where Ct_GI is the average Ct value of either POLG or POLG2, Ct_Actin is the average Ct value for Actin, ΔCt_P are the patient fibroblast values, ΔCt_C are the control fibroblast values, and FC is fold change. Each sample was run five times on a single plate and experiments performed in triplicate.

Real-time quantitative PCR analyses were performed on a TaqMan (Applied Biosystem). PCR reagents were TaqMan Universal PCR Master Mix No Amperase UNG and TaqMan Gene Expression Assay-On-Demand^™ (Applied Biosystem). The initial step of PCR was a 10min hold at 95°C followed by 40 cycles of PCR amplification. PCR reactions were performed in triplicate in 96-wells plates. Analyses were performed using standard curves obtained from hMSCs cDNA with Hmbs (Hs00609297_m1) as the normalizing endogenous control. Fold change relative was calculated based on the 2^(–ΔΔCt) method. Pre-designed TaqMan gene expression assays from Applied Biosystems were: CR: Clock (Hs00231857_m1), Bmal1 (Hs00154147_m1), Per1 (Hs00242988_m1), Per2 (Hs00256143_m1), GSK-3β (Hs01047719_m1); Osteogenic differentiation: Alkaline Phosphatase (Hs01029144_m1), Osteocalcin (Hs00609452_g1), Runx2 (Hs00231692_m1); Adipogenic differentiation: Fabp4 (Hs01086177_m1), Pparγ (Hs01115513_m1), C/Ebpα (Hs00269972_s1) and Gapdh (Hs99999905_m1).