Computational protocol: Epigenetic Modulation of Gene Expression during Keratinocyte Differentiation

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Protocol publication

[…] Total RNA was isolated from epidermal fragments using an Easyblue RNA extraction kit (Intron, Daejeon, Korea) according to the manufacturer's recommended protocol. Two µg of total RNAs was reverse transcribed using moloney-murine leukaemia virus reverse transcriptase (ELPIS Biotech, Daejeon, Korea). Aliquots of an RT mixture were subjected to PCR cycles with a specific primer set. All primers used in this study were designed using the Primer 3 input (http://frodo.wi.mit.edu/primer3/) and are as follows: keratin 14 (K14) 5'-CAGTTCACCTCCTCCAGCTC and 5'-TCCTCAGGTCCTCAATGGTC, keratin 10 (K10) 5'-CTACTCTTCCTCCCGCAGTG and 5'-TTGCCATGCTTTTCATACCA, cyclophilin 5'-CTCCTTTGAGCTGTTTGCAG and 5'-CACCACATGCTTGCCATCCA. PCR fragments were electrophoresed on 1.0% agarose gel and subsequently visualized using ethidium bromide staining. [...] Hybridization images were analyzed using an Agilent DNA Microarray Scanner (Agilent Technology) and data quantification was performed using Agilent Feature Extraction software (Agilent Technology). Preprocessing of raw data and normalization steps were performed using GeneSpring 7.3.1 (Agilent Technology). The individual CpG methylation difference was directly compared based on ratios of signals from control sample DNA. […]

Pipeline specifications

Software tools Primer3, Agilent Feature Extraction, GeneSpring GX
Applications Gene expression microarray analysis, qPCR
Chemicals Eosine Yellowish-(YS), Hematoxylin