6 protocols
Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:
GeneChips were scanned using the Axon Scanner.
(Parameters: Scanning hardware = Axon- GenePix4000B, Scanning software = GenePix [Axon Instruments])
Muscle biopsies were performed prior to any established diagnosis of LPIN1 disease for the 3 patients. Informed consent was obtained from the patients or their families, in agreement with the local ethical committee of Necker Hospital. Human primary myoblasts were obtained as described (68). CD56+ myoblasts were isolated by flow cytometry cell sorting using an anti CD56-APC conjugated antibody (Biosciences ref. 555518). Myoblasts were grown in standard conditions in Ham F10 medium supplemented with 20% foetal bovine serum (FBS), 100 U/mL penicillin, 100 ug/mL streptomycin. Myotubes were derived from confluent myoblast cultures after replacing HamF10 medium by DMEM supplemented with 1% horse serum, 100ug/mL human apotransferrine, 10 ug/mL insulin, and myotubes were used after 10 days of differentiation. Control human myoblasts were kindly provided by Banque de Cellules Cochin-AP-HP (Pr Chelly) and Dr. Djouadi (Inserm U747, Paris). Muscle biopsies were also immediately frozen and kept in liquid nitrogen.
(Parameters: time unit = seconds, temperature unit = C)
After fragmentation, cDNA is end labelled with biotin using Terminal Transferase (using the WT terminal labelling kit of Affymetrix). cDNA is then hybridized to GeneChip(r) human U133 +2.0 Array (Affymetrix).
(Parameters: Amplification = none, Mass unit = Micro gram)
Total RNA was extracted from skeletal muscle samples, myoblasts and myotubes using the Trizol reagent (Invitrogen Life Technologies) and RNeasy Mini Kit (Qiagen). Single-stranded cDNA was synthesized from 2ug of total RNA using the High Capacity RNA-to-cDNA Kit (Applied biosystems). Real-time quantitative PCR (RT-qPCR) was performed using an ABI PRISM 7000 Sequence Detection System instrument and TaqMan Universal PCR Master Mix. Reactions were performed in triplicate using commercially available Taqman kits (Applied Biosystems). Target gene expression was normalized to POLR2A mRNA level using corresponding kits from the same supplier.
(Parameters: Extracted product = total_RNA, Amplification = none)
Myoblasts were subjected to various stress conditions to mimick those believed to trigger episodes of rhabdomyolysis, such as glucose-free medium, high temperature (41 degrees C), pro-inflammatory cytokines (TNF-alpha, IL-1-beta, R&D Systems) and (Poly(I:C) a synthetic inducer of the antiviral response, alone or combined and for various periods of time. The two proinflammatory conditions were retained for further investigations i.e. the association of TNF-alpha and IL-1-beta (10 ng/mL each), and Poly(I:C) (25 ug/mL, Invivogen). 50% confluent cell cultures were synchronized by 12h serum starvation before being submitted to the stress in regular HamF10 medium for the indicated times. Cells were trypsinized and immediately used or stored at -80 degrees C before being processed. All experiments were repeated at least three times. The efficacy of these pro-inflammatory stimulations was assessed by measuring IL6 release in culture medium with an immunoradiometric assay kit (Immunotopics) as previously described (69). Cytokine inhibitors anakinra (inhibitor of IL-1-beta receptor, 1ug/mL) and ab9635 (inhibitor of TNF-alpha, 1ug/mL), or the synthetic glucocorticoid dexamethasone (0.2uM, Dex), were further added in the culture medium respectively 1 hour or 12 hours prior to the 24h-incubation with TNF-alpha + IL-1-beta.