Computational protocol: Bone morphogenetic protein signalling suppresses wound-induced skin repair by inhibiting keratinocyte proliferation and migration

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Protocol publication

[…] For microarray analysis, total RNA was isolated from primary epidermal keratinocytes of P20 WT and TG mice using RNeasy kit (Qiagen, UK), and processed for one-round RNA amplification using RiboAmp RNA Amplification Kit (Molecular Devices, USA). Gene expression array analysis was performed by Mogene LLC (St. Louis, MO, USA) using 44K Whole Mouse Genome 60-mer oligo-microarray (manufactured by Agilent Technologies). Functional annotation of the overrepresented and underrepresented genes was performed as described before (; ) using the NIA Array Analysis software (, and enrichment of the genes in different functional categories was assessed by using the hypergeometric or Fisher’s exact tests. Microarray data has been deposited to the Gene Expression Omnibus (GEO). For qRT-PCR, total RNA was isolated from snap-frozen samples of full-thickness wounds using TRIzol (Invitrogen, UK) () followed by conversion into cDNA using Reverse Transcription System (Promega, UK). PCR primers were designed with Beacon Designer software (Premier Biosoft, Palo Alto; ). qRT-PCR was performed on MyiQ single-colour real-time PCR detection system (Bio-Rad, UK) using SYBR Green master mix (Applied Biosystems, UK). Differences between samples and controls were calculated using the Genex database software (Bio-Rad, UK) based on the Ct (ΔΔCt) equitation method and normalized to Gapdh. Data from triplicates was pooled and statistical analysis was performed using unpaired Student’s t test. […]

Pipeline specifications

Software tools NIA Array Analysis, Beacon Designer
Databases GEO
Applications Gene expression microarray analysis, qPCR
Organisms Mus musculus
Diseases Machado-Joseph Disease