Control plates were harvested for RNA extraction, and the remaining plates were serum starved for 48 (SS48) or 72 hours (SS72) in serum-free basal media (SmBM) to induce growth arrest and expression of SMC differentiation genes, according to conventional protocols. Samples included cells from two separate lots, as well as two technical replicates/vial/time point.
Growth protocol
Human aortic SMCs (Lonza Walkersville Inc., Walkersville, MD, passage #3) were propagated in growth media SmGM-2 with SingleQuot supplements and growth factors (Lonza) in 5% FBS per manufacturer’s instructions.
Extracted molecule
total RNA
Extraction protocol
Cell culture and homogenized aortic samples were harvested with QIAzol Lysis Reagent. Total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Valencia, CA) per standard methods. RNA was quantitated by Nanodrop, and RNA and miRNA quality and integrity were verified using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA). RNA Integrity Numbers (RIN) for all samples ranged from 8.5 to 10.
Label
Cy3
Label protocol
Per manufacturer’s protocol, 100 ng of total RNA per sample was dephosphorylated using calf intestine alkaline phosphatase at 37° C for 30 min, then denatured in DMSO at 100° C for 5 min and placed in an ice-water bath. Ligation to Cyanine 3-pCp was performed using T4 Ligase at 16° C for 2 hours, and labeled miRNA samples were purified using Micro Bio-spin 6 columns.
Hybridization protocol
GE Blocking agent and Hi-RPM hybridization buffer were added to the samples, which were incubated at 100° C for 5 min, and transferred to an ice-water bath. Samples and arrays were then loaded using Agilent SureHyb chambers and gaskets, hybridized at 55° C for 20 hours, and washed (Gene Expression wash buffers 1 and 2).
Scan protocol
Arrays were scanned using an Agilent Microarray Scanner and Feature Extractor (software v 9.5.1).
Description
SS72_3
Data processing
The Sample data were normalized, background subtracted and processed using the Feature Extractor. However, replicates of the same miRNA were not averaged, a process that occurs when the data is properly uploaded to Genespring. Control Flagged spots have been removed.
QC reports were generated by the Feature Extraction software, and the array data were analyzed with Genespring GX 10.0.2 (Agilent). Arrays were examined with Quality Control metrics, principal components analysis (PCA), and hierachical agglomerative clustering (HAC). One chip (6 arrays) clustered separately and was therefore excluded from further analysis. Two additional arrays were excluded for QC reasons. The remaining 10 arrays were subjected to one-way ANOVA at a cutoff of p < 0.05, with Benjamini-Hochberg correction for multiple testing. Significant miRNAs were required to show > 2.0-fold expression change from Control.
