Computational protocol: The Effect of Chondroitin Sulphate and Hyaluronic Acid on Chondrocytes Cultured within a Fibrin-Alginate Hydrogel

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Protocol publication

[…] Cells released from solubilized hydrogels were lysed in TRIzol® LS reagent, as detailed in . The lysate was centrifuged (10 min, 12,000 g, 4 °C) to pellet any insoluble debris. The supernatant was mixed with 80 µL of chloroform, incubated at room temperature for 5 min, then centrifuged (15 min, 12,000 g, 4 °C) to separate the aqueous and organic phases. The upper aqueous phase was drawn off and mixed with an equal volume of 70% ethanol. This solution was then added to a HiBind RNA column from an E.Z.N.A™ Total RNA Kit (Omega, BioTek, Norcross, GA, USA), and the manufacturer’s protocol for RNA isolation was followed.After RNA isolation, the A260/A280 ratio was measured to check for phenol or protein contamination. Then, cDNA was synthesized from this RNA using the QuantiTect® Reverse Transcription Kit (Qiagen, Toronto, Canada) following the manufacturer’s protocol. The cDNA was used as a template for real-time quantitative polymerase chain reaction (qPCR) using a Fermentas Maxima® SYBR Green reagents system (Thermo Fisher Scientific, Ottawa, Canada) in accordance with the manufacturer’s protocol. Amplifications were performed on an MJ Mini thermal cycler equipped with a Bio-Rad MiniOpticon fluorescence detection module. The PCR cycle parameters were 95 °C for 15 s, 65.8 °C for 30 s and 72 °C for 30 s. Expression levels of four types of gene transcripts were analyzed: the mRNAs that code for collagen type I (col1α1), collagen type II (col2α1), aggrecan and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene. The sequences of all qPCR primers used are described in .For each gene, a temperature gradient and serial dilution were run to determine the optimum PCR annealing temperature and PCR amplification efficiency, respectively. To ensure that each primer pair only amplified its specific target gene sequence, both gel electrophoresis and melt-curve analysis were performed on the PCR products. No template controls (NTCs; i.e., PCR reaction mixtures lacking a cDNA template) were run alongside the samples to check for the amplification of primer-dimers or contamination of the reagents. In addition, 6 samples were randomly chosen for “no reverse transcriptase controls” (NRTs) to check for genomic DNA contamination in the isolated RNA. For both the NTCs and NRTs, if amplification took place more than 5 cycles after the lowest expressed transcript in the experimental samples, it was considered negligible, as recommended by Bustin et al. []. Finally, 6 randomly chosen samples were analyzed on a Bio-Rad Experion microfluidic electrophoresis system to verify the integrity of the input RNA used for cDNA synthesis and qPCR amplification. [...] For both the DMMB and the Hoechst assays, a mixed-effects linear model, with the unsupplemented hydrogels as the intercept, was used to identify statistical interactions between the HA and CS treatments that would be indicative of additive, synergistic or antagonistic biological effects. In addition, this allowed for gels that were fabricated at equivalent time points following component mixing to be blocked together in the statistical analysis in order to better resolve significant differences between the non-supplemented hydrogels and the HA, CS and HACS treatment groups.Analysis of variance (ANOVA) together with the Tukey’s honest significant differences post hoctest was used to determine the change in total cellular DNA in untreated hydrogel cultures over the 2-week period of culture All of the above parametric tests were performed using R© (version 2.10.1) software [].The Relative Expression Software Tool (REST) [] was used for the non-parametric analysis of qPCR data on collagen I, collagen II aggrecan and GAPDH gene transcript levels in hydrogel cultures, monolayer cultures and freshly isolated chondrocytes. […]

Pipeline specifications

Software tools e-PCR, REST
Application qPCR
Diseases Osteoarthritis
Chemicals Hyaluronic Acid