AB SOLiD System sequencing; Equine cartilage and bone miRNA repertoires
Equine cartilage and bone miRNA repertoires
Samples were collected on the same location in the middle of the trochlea to avoid technical bias due to sample location in the joint. Explants including epiphyseal cartilage and about 5 mm of the soft underlying bone (considered as subchondral bone) were washed in PBS and placed in DMEM culture medium for subsequent mechanical-loading experiments or flash frozen in liquid nitrogen for subsequent RNA-seq analysis. Harvested cartilage explants were placed into individual compression wells of Biopress culture plates (Flexercell International) in 1.5 ml of culture medium (DMEM, containing penicillin-streptomycin 1%, glutamine 2%, albumin 0.1% and Hepes 30 mM). All experiments were performed at 37C, under ambient atmosphere. A compressive stress was applied to individual samples by the Flexercell FX-4000C system (sinusoidal waveform at 0.5 Hz, 1MPa, 24h00) whereas the control explants stayed in unloaded conditions. Protaglandin E2 (PGE2) production was measured in the media by a high sensitivity commercially available enzyme immunoassay kit (Cayman Chemical). Total RNA was purified from bone using the miRNeasy Mini kit (Qiagen) and the RNeasy MinElute Cleanup kit (Qiagen) for enrichment of small RNA (<200 nt) following the manufacturers protocol. Total RNA quality was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies) loaded with a RNA 6000 Nano Kit. The concentrations of smRNA were determined using a NanoDrop ND-1000 Spectrophotometer and the size and purity were determined using the Bioanalyzer 2100 loaded with a RNA 6000 Nano Kit. SOLiD Total RNA-Seq Kit (Applied Biosystems, Life Technologies). Small RNAs were hybridized and ligated with SOLiD Adaptor Mix, a reverse transcription was performed and the cDNAs were purified using the MinElute PCR purification kit (Qiagen). The 60-80 base pair fraction of the cDNA was excised from a 10% acrylamide gel stained with SYBR Gold (Invitrogen, Life Technologies) and an amplification was realized by PCR (95C - 1min, 18 x (95C -30s, 62C, 30s, 72C, 30s), 72C, 7min) using the SOLiD 5 prime PCR primer and a 3 prime library-specific barcoded SOLiD primer. The amplified DNA was purified using the Purelink PCR micro kit (Invitrogen, Life Technologies) and the 110-130 bp region was excised from a 6% acrylamide gel stained with SYBR Gold (Invitrogen, Life Technologies). The yield and size distribution of the amplified DNA were assessed and pools of three different horses were made to decrease the effect of individual variability.
Experimental Factor: disease
osteochondrosis
Experimental Factor: growth condition
compressed
Experimental Factor: organism part
median femoral trochlea from bone