Accupower rt premix

The RNA was isolated from hDFs and hDF-dcNSCs using the PureLink RNA Mini Kit (Invitrogen). The cDNA was synthesized using AccuPower RT PreMix (Bioneer). The gDNA was isolated from hNPCDFs using the AccuPrep Genomic DNA Extraction Kit (Bioneer). PCR amplification was performed in a GeneTouch Thermal Cycler (Bioer, Hangzhou, China). The resulting PCR products were separated by gel electrophoresis (Clontech) and purified using the MEGAquick-spin Plus Total Fragment DNA Purification Kit (iNtRON Biotechnology). Sequencing of purified PCR products were performed by Macrogen (Seoul, South Korea) using the primer, which was used in the original PCR. For in-depth genotyping, PCR products were cloned using the TOPcloner TA Kit (Enzynomics, Daejeon, South Korea) and transformed into chemically competent DH5α Escherichia coli (Enzynomics) using the heat-shock method. Transformed E. coli cells were spread on Luria-Bertani (LB) Agar LOP plates (Narae Biotech, Gyeonggi-do, South Korea) and incubated overnight at 37°C. On the next day, 10 colonies were inoculated into LB broth (Thermo Fisher Scientific) and incubated overnight at 37°C. Plasmids were isolated from the cultured E. coli using an AccuPrep Plasmid Mini Extraction Kit (Bioneer), and sequencing was performed by Macrogen.

After 21 days, each IPFP-ASCs seeded scaffold was homogenized under liquid nitrogen using a mortar. RNA was isolated from the samples by using RNX-Plus (Sinaclon, IRAN) according to the manufacturer’s instructions. The concentration of RNA was estimated spectrophotometrically with NanoDrop 1000 Spectrophotometer (Wilmington, DE, USA) at A_260/280. The RNA samples were reverse transcribed into first-strand cDNA using the AccuPower^® RT PreMix (Bioneer). Real Time-PCR reactions were performed using the SYBRGreen PCR Mastermix (Applied Biosystems, USA), according to the manufacturer’s instructions. These gene primers were purchased from (Bioneer). The cartilage-specific oligonucleotide primers which were used, were aggrecan (forward 5’-AGGGCGAGTGGAAT-GATGTT-3’; reverse 5’-GGTGGCTGTGCCCTTTTTAC-3’), and collagen type 2a1 (forward 5’-ATGCCACA-CTCAAGTCCCTCAA-3’; reverse 5’-CGCAAGTCTCG-CCAGTCTCC-3’), (IHH) (forward 5’-CACTTCTGCC-TGGTCCTGTT-3’; reverse 5’-GGGCTGAACTGCTTG-TAGG-3’), housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5’-CAAGATCATCAGCAATGCCTCC-3’; reverse, 5’-GC-CA-TCACGCCACAGTTTCC-3’). All experiments were performed in triplicate for each sample. Interpreta-tion of the results was performed using the Pfaffle method and the CT values were normalized with respect to GAPDH expression.

Harvested tissues from the small intestine were immediately snap-frozen and stored at −80°C until RNA extraction was performed. Total RNA was isolated from intestinal tissues and Caco-2 cells using the TRIzol reagent (Invitrogen). cDNA was synthesized using the AccuPower RT premix (Bioneer, Daejeon, Korea) according to the manufacturer's protocol. Real-time RT-PCR was performed using a LightCycler 480 system (Roche, Basel, Switzerland). The primer sequences are provided in Supplementary Table 1. The expression levels of each target gene, determined using LightCycler 480 system software (Roche), were normalized to those of glyceraldehyde 3-phosphate dehydrogenase. Cycle threshold values were used to calculate relative mRNA expression using the 2^−∆∆Ct method.

Total RNA was extracted from RKO and HCT116 cells using the TRIzol reagent (Invitrogen) and treated with DNase I (New England Biolabs). cDNA was synthesized from (1 μg) of total RNA using AccuPower RT PreMix (Bioneer, Daejeon, Republic of Korea), and then amplified by PCR using the appropriate primers for Glut1 (glucose transporter 1), PDK1 (pyruvate dehydrogenase kinase 1), PGK1 (phosphoglycerate kinase 1), CA9 (carbonic anhydrase 9), HIF-1α, and 18S rDNA (Bioneer). qPCR and analyses were performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad).