VALUE = Normalized, log2 ratio (Cy3/Cy5) representing sample/common_reference
The raw pixel intensity images were analyzed using IPLab image processing software package (Scanalytics, Fairfax, VA). The program performs statistical methods that have been previously described to locate specific miRNAs on the array, measure local background for each of them, and subtract the respective background from the spot intensity value (average of triplicate spots). Besides the background subtraction, the IPLab program was also used for within-array normalization and data filtering. Fluorescence ratios within the array were normalized according to a ratio distribution method at confidence level = 99.00. The filtered data from the IPLab program were then uploaded into R version 2.6.1 software package to perform array normalization across all of the samples based upon quantile-quantile (Q-Q) plots, using a procedure known as quantile normalization. After normalization, 1,237 miRNAs were detectable above background.
LCL derived from peripheral lymphocytes of 14 male subjects were obtained from the Autism Genetic Resource Exchange (AGRE, Los Angeles, CA). The subjects included three pairs of monozygotic twins discordant for diagnosis of autism, a normal sibling for 2 of the twin pairs, two pairs of autistic and unaffected siblings, and a pair of normal monozygotic twins. These cell lines had all been used previously for gene expression profiling and thus allowed us to compare miRNA expression profiles with mRNA expression levels across the affected and control samples from both studies. The frozen cells were cultured in L-Glutamine-added RPMI 1640 (Mediatech Inc., Herndon, VA) with 15% triple-0.1 μm-filtered fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin-streptomycin-amphotericin (Mediatech). According to the protocol from the Rutgers University Cell and DNA Repository (which contains the AGRE samples), cultures were split 1:2 every three to four days, and cells were harvested for miRNA isolation three days after a split, while the cell lines were in logarithmic growth phase. All cell lines were cultured and harvested at the same time with the same procedures and reagents to minimize the differences in miRNA expression that might occur as a result of different cell and miRNA preparations.
Hybridization was performed according to the mirVana™ miRNA Probe Set protocol (Ambion).
LCLs were disrupted in TRIzol Reagent (Invitrogen, Carlsbad, CA) and miRNAs were then extracted from the TRIzol lysate using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturers’ protocols. Briefly, ethanol (100%) was added to TRIzol-extracted, purified RNA in water to bring the samples to 25% ethanol and the mixture was then passed through the mirVana glass-fiber filter which allowed passage of small RNA in the filtrate. Ethanol was added to the filtrate to increase the ethanol concentration to 55%, and the mixture was passed through the second glass-fiber filter which immobilized the small RNAs. After washing, the immobilized small RNAs were eluted in DNase-RNase-free water (Invitrogen), yielding an RNA fraction highly enriched in small RNA species ( 200 nt). The concentration of the small RNAs in the final fraction was then measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). To enable comparison of miRNA expression patterns across all of the samples, equal amounts of miRNAs from unaffected siblings and normal control individuals were pooled to make a common reference miRNA that was co-hybridized with each sample on the miRNA microarray.
The microarrays were scanned with a ScanArray 5000 fluorescence scanner (PerkinElmer, Waltham, MA).
miRNA labeling and microarray hybridization were performed using Ambion’s miRNA Labeling Kit and Bioarray Essential Kit, respectively, according to the manufacturer’s instructions. Briefly, a 20-50-nucleotide tail was added to the 3’end of each miRNA in the sample using E.coli Poly (A) Polymerase. The amine-modified miRNAs were then purified and coupled to amine-reactive NHS-ester CyDye fluors (Amersham Biosciences, Piscataway, NJ). A reference design was used for microarray hybridization in this study. The sample miRNAs were coupled with Cy3, whereas the common reference miRNA was coupled with Cy5, and two-colored miRNA microarray analyses were carried out by cohybridizing an equal amount of both miRNA samples onto one slide.