Total RNA including mRNA and miRNA were extracted using the miRNAeasy Mini Kit (Qiagen, Hilden, Germany) following manufacturer`s instructions. An additional DNA digestion step on the RNA binding silica gel membrane of the spin column was performed with DNase I. RNA concentration and the absorbance ratio of 260 nm to 280 nm were measured on the NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of isolated RNA was determined by the RNA integrity number (RIN) with an Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
Samples were labeled using the miRNA Complete Labelling and Hyb Kit from Agilent Technologies. In brief, 100 ng total RNA was dephosphorylated with Calf Intestine Alkaline Phosphatase, denaturated with DMSO and used as RNA acceptor in a T4 RNA ligase mediated reaction with the RNA donor 3’, 5’-cytidine bisphosphate (Cy3-pCp). The labeling reaction was column purified, dried down using a speed-vac at 45°C and resuspended in blocking and hybridization buffer.
Hybridization protocol
Labeled samples were hybridized on human catalog 8-plex 15K miRNA microarrays (Agilent Technologies) according to the supplier’s protocol.
Scan protocol
Microarrays were washed and subsequently scanned at 5 µm resolution with DNA microarray laser scanner (Agilent Technologies) and processed according to the supplier’s protocol.
Description
mRNA Gene expression corresponds to miRNA Data archived under: GSM881448
Data processing
Expression files were generated using Agilent's Genespring GX software
