FlashTag™ Biotin HSR RNA Labeling Kits

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		Label all samples at one time to avoid day-to-day variations. The labeled samples can be stored at -80°C upto 3 months and successive hybridizations

Downstream tips
Label all samples at one time to avoid day-to-day variations. The labeled samples can be stored at -80°C upto 3 months and successive hybridizations

Publication protocol

RNA isolation, quantification and micro-RNA array
Total RNA from VSMC or MV was isolated using miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA was eluted from the column in RNase-free water and stored at -80°C. Quantification of miRNA was performed at the Center for Genetics in Indiana University School of Medicine using Agilent 2100 Bioanalyzer Small RNA kit.

To perform the arrays, total RNA samples were labeled using the Genisphere FlashTag HSR kit. The labeled samples were individually hybridized to Affymetrix GeneChip miRNA 3.0 arrays. They were stained and washed using the standard miRNA protocol. Affymetrix GeneChip Command Console Software (AGCC) was used to scan the arrays and generate CEL files. CEL files were imported into Partek Genomics Suite (Partek, Inc., St. Louis, Mo). RMA (robust multi-array average) signals were generated for all probe sets using the RMA background correction, quantile normalization and summarization by Median Polish [10]. Summarized signals for each probe set were log2 transformed. These log transformed signals were used for Principal Components Analysis, hierarchical clustering and signal histograms to determine if there were any outlier arrays. No outliers were detected in this analysis. Untransformed RMA signals were used for fold change calculations. Contrasts were calculated as required. Fold changes were calculated using the untransformed RMA signals. Probe sets with log2 expression levels < 1.0 were considered very close to background. Probe sets with average expression levels < 1.0 were removed before the False Discovery Rate (FDR) was calculated using the Storey method [11].

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Manufacturer protocol

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