FL-Ovation™ cDNA Fluorescent Module
The FL-Ovation™ cDNA Fluorescent Module is designed for fragmentation and labeling of cDNA for analysis on Agilent Dual Mode DNA gene expression arrays. This product provides a robust, rapid, and simple method for fragmentation and labeling of 2.2 µg of cDNA in less than an hour. The input cDNA can be generated from a wide range of sample quality or quantity using the following NuGEN RNA amplification systems: WT-Ovation™ FFPE System (Cat. #3400), the WT-Ovation™ Pico System (Cat. #3300), or the Ovation™ RNA Amplification System V2 (Cat.# 3100).
The FL-Ovation™ cDNA Fluorescent Module provides optimized reagent mixes and a protocol to process 12 cDNA samples with one-color labeling (Cat.# 4300-12) or 12 samples each for two-color labeling (Cat.# 4310-12).
RNA isolated from matched colon tumor and normal samples stored both as fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) tissue for two years was used as input into the WT-Ovation™ FFPE RNA Amplification System. For the fresh frozen samples, 10 ng of total RNA was used as input in quadruplicate amplification reactions, generating ~13 µg amplified cDNA per reaction. For the FFPE samples, 50 ng of total RNA was used as input in quadruplicate reactions, generating ~6 µg cDNA per reaction. For each reaction, 2.2 µg of amplified cDNA was labeled with ULS-Cy3 and fragmented using the FL-Ovation™ Fluorescent cDNA Module. Approximately 825 ng of labeled and fragmented cDNA was hybridized for 40 hours on Agilent Whole Human Genome 4x44K expression arrays according to manufacturer’s instructions. Arrays were scanned and signals extracted using Agilent’s signal extraction software. Processed Cy3 log10 signals from two replicate arrays from the fresh frozen normal colon sample and two replicate arrays from the FFPE normal colon sample were plotted as scatter plots as shown in graphs A and B above. A high degree of sample replicate reproducibility is realized with signal Pearson correlations of R = 0.995 and R = 0.991 for fresh frozen and FFPE RNA samples, respectively. The high degree of replicate reproducibility observed demonstrates that the system may be used to generate high quality, reliable gene expression data using very small and degraded RNA samples on Agilent arrays.
