|Public on Nov 28, 2013
|cell type: Oral keratinocyte
native mir196a expression: high
|All cells were grown in KGM/Greens medium, as per standard protocols, to 60% confluence before trypsinisation
|Total RNA was extracted from cell pellets using RNEasy minikit (Qiagen), before quantificaiton and QC on an Agilent Bioanalyser
|sample RNA together with the internal control transcripts were labelled with Cyanine 3-CTP (Cy3) dye during an amplification reaction using the Agilent Low Input Quick Amp Kit. The generated fluorescent cRNA (complimentary RNA) products were then purified using Qiagen’s RNeasy Mini Kit. Next, the yield of the linearly amplified cRNA and the Cy3 specific activity were quantified using the Microarray Measurement function of the NanoDrop spectrophotometer. To reduce its structure complexity, 600 ng cRNA of each sample were fragmented into approximately 50-200 bases long. The fragmentation step took place at 60 ºC for 30-min using a Fragmentation Mix for 8-pack microarray format.
|25µl of labelled and fragmented cRNA was hybridised onto Aglient Sureprint G3 60K arrays (8 per slide). The slides were placed in a hybridization rotator set to rotate at 10 rpm inside a 65 °C hybridization oven. After 17 hours, hybridized slides were disassembled and washed.
|The arrays were scanned at 3 μm resolution using Agilent C Microarray Scanner pre-set with the default settings for a 8x60K G3 Microarray Format.
|Gene expression in oral keratinocytes
|Agilent Feature Extraction Software (version 10.5) was used to process the generated image files and create exportable data files containing all the parameters, computed statistical analysis, and feature results. The raw data (.txt) file was loaded into Qlucore Omics Explorer software, before normalisation by the 75th percentile shift method as implemented in the program
|Nov 27, 2013
|Last update date
|Nov 28, 2013
|Keith David Hunter
|University of Sheffield
|School of Clinical Dentistry
|The effect of miR196a of overall gene expression in oral keratinocytes