Enzo BioArray™ Single-Round RNA Amplification and Biotin Labeling System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
cDNA templates should be cleaned up using phenol:chloroform followed by ethanol precipitation. For ethanol precipitation, add ammonium acetate to 2.5M, then add 2.5 volumes of absolute ethanol (-20°C).

Upstream tips
cDNA templates should be cleaned up using phenol:chloroform followed by ethanol precipitation. For ethanol precipitation, add ammonium acetate to 2.5M, then add 2.5 volumes of absolute ethanol (-20°C).

Publication protocol

Motor cortex was dissected from frozen coronal blocks using anatomical landmarks, homogenized and total RNA isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) with RNA quality assessed via analysis on an Agilent 2100 Bioanalyzer. Only samples with an RNA integrity number >7.0 were considered for further analysis. The samples were prepared with 2 μg of total RNA using the Enzo BioArray Single-Round RNA Amplification and Biotin Labeling System. Fifteen micrograms of the purified and fragmented aRNA were hybridized to 14 GeneChip RhG Arrays and 14 GeneChip HG-U133 Plus 2.0 Arrays. Image segmentation analysis and generation of DAT files was performed using Microarray Suite 5.0 (MAS5). Segmented images were normalized and log2 transformed using robust multiarray analysis separately for Rhesus and Human arrays (11). Robust multi-array analysis normalized log2 expression levels were utilized for all of our subsequent analyses.

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