Trizol extraction agent

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)].

Total RNA was extracted from the cell/scaffold constructs 5 days postseeding as described previously and was analyzed for the expression of genes relating to osteogenesis and signal transduction.^{4 ,10 (link)} Total RNA isolation was performed with TRIzol extraction agent (Thermo Fisher Scientific, Waltham, MA) as per the manufacturer's protocol with modifications to increase the yield of RNA.^{13 (link)} cDNA was prepared using a Qiagen First Strand cDNA Reverse Transcription Kit (Qiagen). A housekeeping RT^{2 (link)} Profiler array (Qiagen) was used to ensure the quality of isolated RNA.
Expression was then evaluated using Qiagen RT^{2 (link)} Profiler arrays for human osteogenesis (PAHS-026Z) and signal transduction (PAHS-014Z) with 2 μg of total RNA per array with ∼20.8 ng cDNA per PCR, with samples run in triplicate. Relative fold differences in the gene expression and corresponding significance values were generated through Qiagen data center. Expression of cells seeded on scaffold constructs for 5 days was compared to cell monolayers differentiated on polystyrene substrates for 21 days with osteodifferentiation media.^{9 (link)}