Amino allyl arna kit

RNAs (including miRNAs) were extracted using the mirVana™ miRNA Isolation Kit (Life Technologies Japan Ltd.) according to the recommendations of the manufacturer. Microarray analysis was carried out using the Human Oligo chip 25 K (ID QH0ZG35) by Toray Co. (http://www.3d-gene.com/en/about/; Tokyo, Japan) as described previously [9 (link)]. In brief, total RNA was amplified using an Amino Allyl aRNA kit (Ambion). RNA from cells was labeled with Cy3 Mono-Reactive Dye (GE Healthcare Japan Co., Tokyo, Japan), and control RNA was labeled with Cy5 Mono-Reactive Dye (GE Healthcare). After purification, each 1-μg sample was mixed and hybridized at 37 °C for 16 h. After washing, the hybridized chip was scanned using a 3D-Gene Scanner 3000 (Toray Co.). Background was subtracted from the raw data and the values were normalized according to a median Cy3/Cy5 ratio of 1.

For mRNA microarray analysis, 3D-Gene Human Oligo Chip 25 K (Toray Industries, Inc., Tokyo, Japan) was used. Total RNA was amplified using an amino allyl aRNA kit (Ambion, Inc., Foster City, CA, USA). The total RNA was labeled with cyanine 5 (Cy5) using Amersham Cy5 mono-reactive dye (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Cy5-labeled aRNAs were individually hybridized at 37 °C for 16 h. The chips were washed and scanned using a 3D-Gene scanner 3000 (Toray Industries, Inc.) and analyzed using 3D-Gene extraction software (Toray Industries, Inc.). The signals detected for each gene were subjected to global normalization (the median of the detected signal intensity was adjusted to 25).