Impact of high-molecular weight hyaluronic acid on the gene expression of rabbit Achilles tenocytes in vitro
Description
For three rabbit donors (denoted as R5, R8, and R11 in the Excel sheet), we present the impact of high-molecular weight hyaluronic acid (HA; Mw = 1.01 – 1.80 MDa) daily supplementation to Achilles tenocytes in vitro at time points 3 days, 7 days, and 14 days against the control which received no HA, but had otherwise the same conditions. Gene expression of target genes Biglycan, Decorin, Aggrecan, Collagen 1A1, Collagen 1A2, Collagen 3, LOX, IL-6, TNF-alpha, PAR-2, ALOX-15, Tenascin-C, Tenomodulin, Mohawk, alpha-SMA, TIMP 1, MMP 2, MMP 9, and ki67 are presented; the data were normalized to gene expression of corresponding cell culture without HA supplementation.
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At the respective time point, total RNA was isolated using the RNeasy Plus Mini Kit with RNase-free DNase treatment (Qiagen, # 74136, Hilden, Germany), following the manufacturer’s protocol. Amount and purity of RNA was measured with Nanodrop One (ThermoFisher, Basel, Switzerland). For reverse transcription (RT), 500 ng RNA was investigated in a reaction volume of 20 µL (SuperScript III Reverse Tran-scriptase, # 18080085, ThermoFisher, Basel, Switzerland; Oligo(dT)12-18 Primer, # 18418012, ThermoFisher, Basel, Switzerland; RNase Inhibitor, # N8080119, Applied Biosystem, ThermoFisher, Basel, Switzerland; dNTP, # 18427013, Invitrogen, Ther-moFisher, Basel, Switzerland) using a compact thermocycler (Masterscycler personal, Eppendorf, Germany). Real-Time PCR reactions were performed in technical tripli-cates with 4 µl of the resulting cDNA using the Quant Studio 5 (Applied Biosystems, ThermoFisher, Basel, Switzerland) and Fast SYBRTM Green Master Mix (ThermoFisher, # 4385612, Basel, Switzerland). The samples were heated to 95 °C for 3 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 20 s. All primers were synthesized by Micro-synth, Balgach, Switzerland. Relative expression analysis was performed, using the comparative 2-delta deltaCT method with 18S as a reference gene, which was stable over the two conditions analyzed. Results are presented as fold change normalized to control, i.e., compared to samples cultivated without HA (set to 1).
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Funding
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
310030_197578