source tissue: inflammed tonsils cell type: memory B cell
Treatment protocol
Tonsils were not treated prior to extraction.
Growth protocol
Tonsils obtained from tonsillectomy were placed on ice until isolation of mononuclear population.
Extracted molecule
total RNA
Extraction protocol
Tonsils were minced and the mononuclear cell population was isolated from the buffy layer following centrifugation in Ficoll-paque. CD19+ B cells were then isolated using CD19+ immunomagnetic dynabeads (Invitrogen). These CD19+ immunomagnet was removed using CD19 DETACHaBEAD (Invitrogen) and the cells were then stained with CD38-PE and IgD-FITC antibodies (BD Pharmingen). Subsequently, the cells were separated into naive (IgD+, CD38-), germinal center (IgD-, CD38+), and memory (IgD-, CD38-) subsets using FACSAriaII high-speed cell sorter (Becton Dickinson). The cells were pelleted and total RNA was extracted using mirVana miRNA isolation kit (Ambion).
Label
Hy3
Label protocol
Spike-in miRNAs were added to 0.5ug of total RNA of each sample and this mixture was first CIP treated (30 min at 37 C, 5 min at 95 C, and iced for 2 min) before labeling with Hy3 (1 hr at 16 C, 15 min at 65 C, store at 4 C). The labelled reaction was hybridized within 1-2 hrs.
Hybridization protocol
Hybridization buffer was added to each labelled samples and denatured at 95 C for 2 min and then iced for 2 min. The samples were then hybridized to miRCURY LNA v.11 microarray at 56 C for 16 h in agilent hybridization oven rotated at 20 RPM. The following day, the slides were washed, dried, and scanned immediately.
Scan protocol
GenePix personal 4100A scanner (Axon Instruments) was used to scan the microarray at the following settings, wavelength: 532nm, PMT gain: 700, scan resolution: 10 microns. The GenePix Array List (GAL) file, based on the array lot number, were downloaded from the EXIQON website.
Data processing
The threshold signal was calculated to be the mean of negative control spots+3 standard deviations; and all signals below this threshold were deleted. Data quality of each microarray was evaluated using the the coefficient of variation of negative controls, positive controls, and spike-in controls. The signals across samples were normalized using a factor derived from the mean of the signal intensity of several house-keeping small RNAs (U6, snRNA, snoRNAs, and 5SRNAs). Prior to hierarchical clustering and heat map construction, the normalized data was log2 transformed, and data filtered to include only the top 50% of miRNAs with the highest level of standard deviation across samples. The MultiExperiment Viewer software v4 (http://mev.tm4.org) was used for data analysis and viewing.
