Ovation® RNA Amplification System V2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always keep thawed reagents and reaction tubes on ice	Do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Upstream tips
Always keep thawed reagents and reaction tubes on ice
Protocol tips
Do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Publication protocol

Fifty nanograms of total RNA were converted by Single Primer Isothermal Amplification (SPIA®) to cDNA using the Ovation RNA Amplification System v2 according to the manufacturer’s recommended protocol. The amplified SPIA® cDNA product was purified through a QIAGEN QIAquick PCR purification column according to modifications recommended by NuGEN. A sample (3.75 ug) of cDNA was fragmented (average fragment size = 85 bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module v2 per the manufacturer’s recommended protocol. The resulting biotin-labeled cDNA was mixed with Affymetrix eukaryotic hybridization buffer, placed onto Affymetrix GeneChip Bovine Expression arrays, and incubated at 45 °C for 18 h at 60 rpm in an Affymetrix Model 640 Genechip Hybridization Oven (Affymetrix, Inc., Santa Clara, CA). Following hybridization, the arrays were washed, stained with streptavidin-phycoerythrin, and the signal was amplified with anti-streptavidin antibody using the Affymetrix Model 450 Fluidics Station. Arrays were scanned with an Affymetrix Model 3000 scanner with 7G upgrade, and data were collected using the GeneChip operating software (GCOS) v1.4. Expression value estimates were generated using the Microarray Analysis Suite (MAS) v5.0 software.

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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