Microvesicular mRNA profiling of SW480 human colorectal adenocarcinoma cells

We analyzed mRNA profile of SW480-derived microvesicles using microarray Keywords: Microvesicular mRNA profiling The total RNA was extracted from purified microvesicles derived from SW480 by the single-step isolation method using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s protocol. For microarray hybridizations, we followed protocol described in the instruction manual of Illumina TotalPrep RNA Amplification kit (Ambion, Austin, TX). In brief, 640 ng of total RNA from microvesicles was used to synthesize single-stranded cDNA using T7-Oligo(dT) promoter primer and RNase H-facilitated second-strand cDNA, which was purified and served as a template in the subsequent in vitro transcription. The in vitro transcription was carried out in the presence of T7 RNA polymerase and a biotinylated NTP mixture for cRNA. There are four replicate hybridizations. Same amount (Each 1.5 μg) of cRNA was hybridized to arrays, and subsequently labeled with Cy3-streptavidin (Amersham Biosciences, Little Chalfont, UK) and scanned with a Bead Station (Illumina, San Diego, CA).