Omniscript reverse transcriptase

Primers were designed using an online primer design program [14 (link)] (Table 1). Total RNA was isolated and cDNA prepared as described previously [13 (link)]. Briefly, total RNA was isolated in C2C12 cells (2 × 10⁴ cells/well in a 24-well plate) using TRIzol reagent (Life Technologies, MD, USA) and the first strand cDNA synthesized using 2 μg of total RNA, 1 μM of oligo-dT₁₈ primer, and Omniscript Reverse Transcriptase (Qiagen, CA, USA). SYBR green-based quantitative PCR was performed using the Stratagene Mx3000P Real-Time PCR system and Brilliant SYBR Green Master Mix (Stratagene, CA, USA) as described previously [13 (link)]. All reactions were run in triplicate and data analyzed using the 2^−ΔΔCT method [15 (link),16 (link)]. GAPDH was used as the control gene. Significance was determined with GAPDH-normalized 2^−ΔΔCT values. The mRNA levels in zebrafish were evaluated as follows. Five days after fertilization, 20 embryos per well were transferred to a 12-well plate with 2 ml of test solution. After 1 day, total RNA was isolated using TRIzol reagent and the first strand cDNA synthesized using 2 μg of total RNA, 1 μM of oligo-dT₁₈ primer, and Omniscript Reverse Transcriptase (Qiagen, CA, USA). SYBR green-based quantitative PCR was performed as described above.

The transdifferentiation assay was performed as described before.^{41 (link)} Briefly, ATDC5 cells were cultured in chondrogenic differentiation medium for 7 days and thereafter cultured in osteogenic differentiation medium containing 50 ng/mL recombinant human Ctgf (PeproTech Inc., Rocky Hill, NJ, USA). Cells were harvested after 24 and 48 h of osteogenic differentiation, respectively. RNA from cell lysates was isolated using the RNeasy Mini Kit and cDNA was generated using Omniscript Reverse Transcriptase (both Qiagen). qRT-PCR expression analysis was performed using Platinum™ SYBR™ Green qPCR SuperMix-UDG and ROX™ Reference Dye (both Thermo Fisher Scientific). The primer sequences of the analyzed genes are shown in Table S1. B2m expression was used as the reference housekeeping gene. Data analysis was performed according to the delta-delta comparative threshold cycle (2^-ΔΔCT) method, and results are shown as fold-change expression values relative to controls.

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of total RNA were calculated using a NanoDrop Spectrophotometer at 260 and 280 nm (Thermo Fisher Scientific, Waltham, MA, USA). The first cDNA strand was synthesized with 2 μg of total RNA and 1 μM of Oligo-dT₁₈ primer using Omniscript Reverse Transcriptase (Qiagen, Valencia, CA, USA). Quantitative real-time PCR (qRT-PCR) amplification was performed using an S1000 thermal cycler real-time PCR system and iQ SYBR Green supermix (Bio-Rad, Hercules, CA, USA) in the presence of first-strand cDNA (1:25 dilution) and 20 pmol of primers according to the manufacturer’s protocols. The primers used to amplify specific products are listed in Table 1. All reactions were run in triplicate and data were analyzed by the 2^−ΔΔCT method. Significance was determined by a two-tailed Student's t-test (*p<0.05, **p<0.01, and ***p< 0.001).