Cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 250 ng/ml Fungizone (Amphotericin B; Invitrogen) and 600 IU of IL-2. Every three days, the cells were fed with fresh media supplemented with 600 IU of IL-2.
Extracted molecule
total RNA
Extraction protocol
miRNeasy
Label
Cy3
Label protocol
The microarray assay was performed using a service provider (LC Sciences). The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore), and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent Cy3 dye staining.
Hybridization protocol
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
Scan protocol
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
Data processing
Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix. Normalization is carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data. The normalization is to remove system-related variations, such as sample amount variations, different labeling dyes, and signal gain differences of scanners so that biological variations can be faithfully revealed [B. M. Bolstad, R. A. Irizarry, M. Astrandand T. P. Speed, (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias, Bioinformatics, 19 (2), 185-193]. A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5. CV is calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. A t-test is performed between SNK6 and “SNT16” samples using the probe repeats as individual samples. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using hierarchical method and is performed with average linkage and Euclidean distance metric. All data processes, except clustering plot, are carried out using in-house developed computer programs. The clustering plot is generated using TIGR MeV (Multiple Experimental Viewer) software from The Institute for Genomic Research.
