Scanarray

IgG levels against different human and avian influenza HA types were measured using a protein microarray platform as described previously30 (link)31 (link)32 (link)33 (link)34 (link)35 (link). Briefly, recombinant proteins of the HA1 part of HA of different influenza virus subtypes (see supplementary material S2) were printed onto nitrocellulose-coated glass slides (64pad, Oncyte Avid, Grace Biolabs, Bend, USA) using a non-contact Piezorray spotter (Perkin Elmer, Waltham, USA). Subsequently, dried blood spots were eluted as described previously and samples were tested at a 1:80 dilution30 (link). A Dylight649-labelled goat-anti-human IgG (Fc-fragment specific, Jackson ImmunoResearch) was used to bind to serum antibodies and fluorescence was quantified by means of a microarray scanner (ScanArray, Perkin Elmer). The protein microarray technique allows simultaneous and standardized detection of antibodies against different influenza subtypes in a minute serum quantity. It has also been used to measure influenza IgG titers in humans35 (link).

miRNA expression was evaluated by miRCURY LNA™ microRNA Array (Exiqon), which contains 3100 capture probes covering human, mouse and rat miRNAs. In particular, this microarray analyzes the expression of 1135 mouse miRNAs. 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, and then a wash sequence was performed. Microarray was dried by centrifugation and scanned by a laser scanner (ScanArray, PerkinElmer, Waltham, MA).

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)].