miRNA expression analysis in all individual lung and blood serum specimens was carried out by microarrays (miRCURY LNA™ microRNA Array, Exiqon) containing 3100 capture probes covering human, mouse and rat miRNAs. In particular, this microarray analyzes the expression of 1135 mouse miRNAs. The RNA from each sample was labeled with Label IT® miRNA Labeling Kits, Version 2 (Mirus Bio, WI) following the standard protocol. Total RNA (500 ng) was mixed with 10 μl of 10x labeling buffer, 4 μl Label IT reagent (containing Cy 3 or Cy 5 fluorescent tracers), and water to 86 μl. The samples were incubated at 36°C for 1 h and the reaction was stopped by adding 10 μl Stop Reagent. The samples were purified onto a chromatographic column, and hybridized to the microarray in GlassArray Hybridization Cassettes (Invitrogen Ltd, Paisley, UK) in a water bath at 37°C for 16 h. After a wash sequence, the microarrays were dried by centrifugation and scanned by laser scanner (ScanArray, PerkinElmer, Waltham, MA).
Microarray data were validated by real time-qPCR for miR-146a, miR-191, miR-199b, and miR-223 as previously described [27 (link)].
Microarray data were validated by real time-qPCR for miR-146a, miR-191, miR-199b, and miR-223 as previously described [27 (link)].
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