Cy3 Mono-Reactive Dye Pack

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

Sample amplification, labeling, and microarray processing were performed by the Covance Genomics Laboratory (Seattle, WA). Briefly, samples were amplified using a custom two-cycle RT-IVT amplification protocol. For each sample, 5ng of total RNA was mixed with 250ng of pBR322 (Life Technologies) to act as a carrier. The MessageAmp II aRNA Amplification Kit was utilized for the first round of amplification and the Amino Allyl MessageAmp II aRNA Amplification Kit for the second round of amplification (Life Technologies). Following amplification, 5µg of cRNA was labeled with Cy3 mono-Reactive Dye (GE Healthcare). Each labeled aRNA was resolved using a Bioanalyzer with RNA 6000 Nano kit reagents (Agilent Technologies) before hybridization. Samples were evaluated for yield and size distribution, then normalized to 600ng input, fragmented, and hybridized to Agilent Human 8×60K Arrays. Gene expression data quality was assessed using standard Agilent quality control metrics. To control for batch effects, common RNA pool control samples were amplified and hybridized in each batch. A total of 1,225 samples passed sample quality control (QC), including 1,202 experimental samples and 23 control samples. The data discussed in this publication are accessible through the Allen Brain Atlas data portal (http://www.brain-map.org) or directly at http://www.brainspan.org.

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Manufacturer protocol

Download the product protocol from GE Healthcare Life Sciences for Cy3 Mono-Reactive Dye Pack below.

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