7 protocols
Invariant based normalization as described by Pradervand et al., RNA, 2009
Images generated by the GenePix 4200AL scanner were imported to GenePix 6 microarray analysis software.
Labeled RNA was hybridized to LNA miChip array platforms (Exiqon LNA Array probes V9.2, containing 558 human specific miRNA sequences) over 16 hours at 54°C using a rotational hybridization chamber. Arrays were subsequently washed in varying stringency washes, rinsed, drained and scanned using a Genepix 4200AL laser scanner (Auto Loader, Axon Instruments)
RNA labeling and hybridization on miRNA microarray chips was performed as previously published (Castoldi et al., Nat Protoc. 2008;3(2):321-329).
Normal and malignant plasma cells were purified from bone marrow aspirates using CD138-microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) and purity was assessed by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany). Established human myeloma cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) or the American Type Cell Culture (Wesel, Germany) and cultured as recommended. HG-1 was generated in the Myeloma Research Laboratory Heidelberg, Germany, the XG-lines at INSERM Unit 1040 (Montpellier, France). Cells were lysed in RLT buffer (Qiagen, Hilden, Germany) and stored at -80 degrees C prior to RNA/miRNA extraction.
RNA extraction was performed using the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. The flow-through containing miRNAs was processed using the RNeasy MinElute Cleanup Kit (Qiagen). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Boblingen, Germany).