For reverse transcription polymerase chain reaction (RT-PCR) and quantitative
polymerase chain reaction (qPCR) assay, the cell-seeded hydrogels were incubated
in the chondrocyte maintenance medium for 14 days. Total RNA was extracted using
Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. Total RNA (1 µg) was used for cDNA synthesis with a Superscript
kit (Invitrogen, Carlsbad, CA, USA) with random hexamers. RT-PCR reactions were
conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s.
PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried
out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul,
Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City,
CA, USA) according to the manufacturers’ instructions. The reaction mixture was
made up to 50 µL. The relative transcript quantities were calculated using the
2-ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as the endogenous reference gene amplified from the
samples. The primer sequences used for RT-PCR and qPCR are summarized inTable 1 . The reactions
were run in triplicate in three independent experiments.
polymerase chain reaction (qPCR) assay, the cell-seeded hydrogels were incubated
in the chondrocyte maintenance medium for 14 days. Total RNA was extracted using
Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. Total RNA (1 µg) was used for cDNA synthesis with a Superscript
kit (Invitrogen, Carlsbad, CA, USA) with random hexamers. RT-PCR reactions were
conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s.
PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried
out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul,
Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City,
CA, USA) according to the manufacturers’ instructions. The reaction mixture was
made up to 50 µL. The relative transcript quantities were calculated using the
2-ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as the endogenous reference gene amplified from the
samples. The primer sequences used for RT-PCR and qPCR are summarized in
were run in triplicate in three independent experiments.