Mx3005p real time pcr cycler

TGF-β signaling pathway-related genes were detected by using RT² Profiler PCR Array kits (PASS-235Z, Qiagen, Hilden, Germany) in the Mx3005p real-time PCR cycler (Agilent Technologies, Santa Clara, CA, USA) as described previously [28 (link)]. The mRNA expression levels of aim genes were analyzed using the 2^−ΔΔCt methods by PCR Array system software version 3.5 (Qiagen, Hilden, Germany).

RNA was extracted from both control hMSC cultures, grown on a polystyrene coated tissue culture surface and Gelfoam®-embedded hMSCs at days 7 and 21 of differentiation. Total RNA was isolated using TRIzol extraction agent (Thermo Fisher) as per the manufacturer's protocol and as reported earlier [18 (link)]. Briefly, total RNA was prepared and further purified using a RNeasy mini kit (Qiagen); cDNA was prepared using a high-capacity cDNA reverse transcription kit (Applied Biosystems); and qPCR analysis of the expression of the bone-specific markers osteopontin (OPN) and osteocalcin (OCN) was carried out using SYBR green master mix (Thermo Fisher) with GAPDH serving as the housekeeping gene using MX3005P real-time PCR cycler (Agilent).
Several preliminary experiments were run to determine ideal qPCR protocol, PCR mix, and annealing temperatures. qPCR was run using ABsolute Blue qPCR Mix (Thermo Fisher Scientific), with each reaction comprising of 5.0 μL cDNA solution, 12.5 μL ABsolute Blue SYBR Green ROX, 5.0 μL RNase Free Water, and 2.5 μL of the appropriate primers. All primer sequences and PCR conditions were derived from a previously published report [5 (link)].