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Poly(A) +cDNA was transcribed in vitro by incorporation of biotinylated CTP and UTP (Enzo Diagnostics, Farmingdale, NY) using a BioArray High Yield RNA Transcript Labeling kit according to the manufacturer's instructions.
Total RNA (20 μg) was used to synthesize double-stranded cDNA using Superscript ®Choice System (Life Technologies), and the template for an in vitro transcription reaction was used to synthesize biotin-labeled antisense cRNA (BioArray™ High Yield RNA Transcript Labeling Kit; Enzo Diagnostics, Farmingdale, NY, USA).
Biotinylated cRNA was prepared using the standard Affymetrix protocol [ 8 ] except that the Epicentre AmpliScribe™ T7 polymerase (Epicentre, Madison, WI) was substituted for the ENZO T7 polymerase (BioArray, High Yield RNA Transcript Labeling Kit, ENZO Diagnostics, Inc., Farmingdale, NY) for 2 of the treated and 2 of the untreated pooled samples.
In addition, an antibody against α-actin (Enzo Diagnostics, Inc, Farmingdale, NY) was used to confirm SMCs in the media.