RNA was isolated from cells by using the RNeasy Mini Kit (Qiagen, UK), and each sample was analyzed in triplicate in accordance with the manufacturer’s protocol: 1 ng/μl of extracted RNA was used for cDNA synthesis by using the QuantiTect Reverse Transcription Kit (Qiagen, UK) according to the manufacturer’s protocol.
Inventoried Taqman assays (Applied Biosystems, Warrington, UK) were used for the genes as follows: adipogenesis, fatty acid synthase (FASN), perilipin (PLIN) and peroxisome proliferator-activated receptor gamma (PPARG); chondrogenesis, cartilage oligomeric matrix protein (COMP), aggrecan (ACAN), and Sry-related HMG box 9 (SOX-9); and osteogenesis, bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 6 (BMP6), and osteoclastogenesis inhibitory factor (OPG). Corneal epithelial progenitor genes; hairy and enhancer of split 1 (Drosophila), (HES1), frizzled-related protein (FRZB1), dopachrometautomerase (dopachrome delta-isomerase, tyrosine-related protein 2) (DCT), superoxide dismutase 2, mitochondrial (SOD2), ATP-binding cassette, subfamily G member 2 (ABCG2), E-cadherin (cadherin 1 type 1, CDH1), and keratin 19 (KRT19). Differentiated corneal epithelial genes; keratin 3 (KRT3), keratin 12 (KRT12), keratin 24 (KRT24), desmoglein 3 (DSG3). Housekeeping gene; 18S rRNA.
Amplification was performed by using the Mx2005P multicolor 96-well PCR system (Stratagene, Agilent Technologies, Cheshire, UK) with parameters of 95°C for 10 minutes) followed by 40 cycles of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 30 seconds. Data analysis was performed by using MxPro software, version 4.2 (Stratagene, UK) to measure the threshold cycle (Ct) for each reaction. The mean Ct value was established by using triplicate Ct values, and analysis was completed by using the ΔΔCt method [65 (link)].
Inventoried Taqman assays (Applied Biosystems, Warrington, UK) were used for the genes as follows: adipogenesis, fatty acid synthase (FASN), perilipin (PLIN) and peroxisome proliferator-activated receptor gamma (PPARG); chondrogenesis, cartilage oligomeric matrix protein (COMP), aggrecan (ACAN), and Sry-related HMG box 9 (SOX-9); and osteogenesis, bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 6 (BMP6), and osteoclastogenesis inhibitory factor (OPG). Corneal epithelial progenitor genes; hairy and enhancer of split 1 (Drosophila), (HES1), frizzled-related protein (FRZB1), dopachrometautomerase (dopachrome delta-isomerase, tyrosine-related protein 2) (DCT), superoxide dismutase 2, mitochondrial (SOD2), ATP-binding cassette, subfamily G member 2 (ABCG2), E-cadherin (cadherin 1 type 1, CDH1), and keratin 19 (KRT19). Differentiated corneal epithelial genes; keratin 3 (KRT3), keratin 12 (KRT12), keratin 24 (KRT24), desmoglein 3 (DSG3). Housekeeping gene; 18S rRNA.
Amplification was performed by using the Mx2005P multicolor 96-well PCR system (Stratagene, Agilent Technologies, Cheshire, UK) with parameters of 95°C for 10 minutes) followed by 40 cycles of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 30 seconds. Data analysis was performed by using MxPro software, version 4.2 (Stratagene, UK) to measure the threshold cycle (Ct) for each reaction. The mean Ct value was established by using triplicate Ct values, and analysis was completed by using the ΔΔCt method [65 (link)].
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