All cell populations were isolated on a PercollTM density gradient followed by immunomagnetic depletion as described in Bjerregaard MD, Jurlander J, Klausen P, Borregaard N, Cowland JB: The in vivo profile of transcription factors during neutrophil differentiation in human bone marrow. Blood 2003, 101: 4322-4332.
Label
Hy5
Label protocol
Total RNA extracted from BM and PB cell populations was labeled with the miRCURYTM Array Labeling kit (Exiqon, Vedbæk, Denmark) and hybridized to miRCURYTM LNA microRNA Arrays (Exiqon) in a Tecan HS400 hybridization station (Tecan Nordic AB, Brøndby, Denmark). Labeling with the fluorescent dyes Hy3 and Hy5 and preparation of wash buffers were done according to the manufacturer’s instructions (Exiqon)
Hybridization protocol
Slides were placed in the hybridization chambers and primed by injecting hybridization buffer into each chamber. Finally, the target preparations were injected and incubated for 16 hours at 60ºC with medium agitation. After hybridization, slides were washed, soaked in wash buffers, and dried as recommended by the manufacturer (Exiqon). Slides were removed from the hybridization station and kept in the dark in a nitrogen sphere until scanning.
Scan protocol
Slides were scanned in an Agilent DNA microarray scanner (G2565BA). Image analysis was performed using the Genepix® Pro Software (version 6.1.2; Molecular Devices, Philadelphia, USA). A gene array list(GAL) file (www.exiqon.com. GAL file: 208000V8.1_208001V8.1_208002V8.1_lot_number_20278-20399_hsap.gal. 2-11-2007) with miRNA annotations according to miRBase (version 10) was applied to annotate features and generate a Genepix Results (GPR) File for each image, with median foreground and mean background values for each spot.
GPR files were loaded into the R (version 2.0.1) software environment (Gentleman R, Isaka R: R: A Language for Data Analysis and Graphics. Journal of Computational and Graphical Statistics 1996, 5: 299-314) using the Limma software package (Smyth, G. K. (2005). Limma: linear models for microarray data. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor, R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, W. Huber (eds.), Springer, New York, pages 397–420). Normalization of fluorescence signals between all samples was performed in R (version 2.0.1) using qspline (Workman C, Jensen LJ, Jarmer H, Berka R, Gautier L, Nielser HB et al.: A new non-linear normalization method for reducing variability in DNA microarray experiments. Genome Biol 2002, 3(9):research0048) prior to all statistical analysis.
qspline normalized fluorescence intensities sorted according to increasing p-values as calculated in a one-way ANOVA performed to identify differentially expressed miRNA genes between the bone marrow and peripheral blood cell populations from the 4 donors
