Ovation® RNA Amplification System V2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Protocol tips
do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Publication protocol

Gene expression array analysis
Total RNA was extracted using an AllPrep Micro Kit (Qiagen, Hilden, Germany). Total RNA samples were subjected to gene expression microarray analysis using a SurePrint G3 Human GE Microarray 8x60K Kit (product no. G4851A, design ID 28004, Agilent Technologies, Santa Clara, CA, USA) by following the manufacturer’s instructions. This array contains a total of 50,599 probes covering 27,958 RefSeq genes and 7,419 lincRNAs. Total RNAs (5 ng each) were amplified using Ovation Pico WTA System V2 (NuGen Technologies, San Carlos, CA, USA) and labelled with Cy3 using a Low Input Quick Amp Labeling Kit (Agilent). The resultant cRNAs were fragmented at 60oC for 30 min in the dark, and 600 ng of them were hybridized to the microarray at 65oC for 17 h. After washing, slides were scanned with an Agilent G2565CA Microarray Scanner. Feature Extraction Software (version 10.7.3.1) was used to calculate signal intensities from the scanned images. Raw intensity data were imported into the Subio Platform software (https://www.subio.jp/) and normalized (75th percentile global normalization). The hierarchical clustering analyses were performed using heatmap.2 in the R package gplots (http://cran.r-project.org/web/packages/gplots/gplots.pdf), and hclust (https://stat.ethz.ch/R-manual/R-devel/library/stats/html/hclust.html), with complete linkage with Euclidean distance as parameters. The expression array dataset has been deposited in the NCBI GEO and is accessible through GEO Series accession number GSE73090.

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Manufacturer protocol

Download the product protocol from NuGEN for Ovation® RNA Amplification System V2 below.

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