NuGEN Illumina Protocol
The NuGEN Illumina Solution enables the use of Ovation-generated cDNA for analysis on the Illumina’s Genome-Wide Expression BeadChips. The NuGEN Ovation and WT-Ovation technologies for RNA amplification are ideal for use with small and compromised RNA samples typically obtained from clinical specimens such as whole blood, LCM, sorted cells, fine needle biopsies, and FFPE samples.
The Illumina Solution consists of a labeling protocol described in the Illumina Solution Application Note #2. This Note details the cDNA labeling method, lists the specific materials and reagents required, and provides reagent source and order information.
The workflow consists of the following steps:
- Define Sample Type: Based on sample characteristics, choose one of NuGEN's validated amplification products such as WT-Ovation FFPE System, WT-Ovation Pico System, or the Ovation RNA Amplification System V2.
- Amplify RNA: Obtain the appropriate amplification system and amplify RNA according to NuGEN protocols to yield amplified cDNA.
- Label cDNA: Using the detailed protocol outlined in the NuGEN Illumina Solution Application Note #2, label the amplified cDNA and proceed with hybridization to the Illumina whole genome expression arrays.
The NuGEN Labeling Protocol for Illumina arrays enabes access to challenging samples.
The reproducibility of the WT-Ovation FFPE Solution-generated targets on BeadArray was determined by pairwise Pearson correlations among four independently amplified and labeled replicates for an FFPE sample as well as the matched fresh frozen sample. The average R2 of all pairwise comparisons was 0.99 for fresh frozen and 0.98 for FFPE samples. An example of the linear array signal scatter plots for the FFPE sample is shown above.
