The Ovation Systems provide simple, robust workflows optimized for use with challenging samples down to a single cell. These sample preparation solutions enable analysis on a broad range of genomic platforms, including Next-Gen Sequencing, microarrays, and qPCR.
Working with challenging and precious samples is no longer a barrier to expression profiling. These application-based kits have been designed to enable research with precious clinical samples, such as biopsies, sorted cells, laser capture microdissection, Formalin- Fixed Paraffin- Embedded tissues (FFPE), and whole blood. The Ovation family of products allows you to work with a range of samples:
- Samples as small as 10 picograms total RNA
- Degraded samples with RIN scores as low as 2.0
- Formalin-fixed, paraffin-embedded (FFPE) samples (Figure 1)
- Whole blood
- Direct cell lysate from the equivalent of one or a few cells (Figure 2)
For next-generation sequencing, the Ovation RNA-Seq System V2 extends the power and flexibility of this new technology to sample preparation from as little as 500 picograms of total RNA. (Figures 3 and 4). In addition, more comprehensive representation of the transcriptome can be obtained through elimination of the potential bias introduced by poly(A)+ RNA selection and ribosomal RNA reduction.
Highly concordant differential expression analysis results between FF and FFPE samples
Using the WT-Ovation FFPE System, total RNA isolated from lung cancer and normal FFPE samples (50 ng) as well as matching fresh frozen (FF) tissues (2 ng) were amplified, labeled, and hybridized to Affymetrix GeneChip® HG-U133A Arrays. 815 genes were detected to be highly differentially expressed (signal log ratio, SLR) between the cancer and normal samples. Highly concordant results are demonstrated between FF and FFPE samples.
Clustering of microarray data obtained with the WT-Ovation One Direct System from cell lysates of 10 cells
In order to demonstrate array performance with cell lysates, triplicate amplifications were performed representing 10-cell samples of untreated THP-1 cells (red), PMA treated/serum starved cells (green), and PMA treated/serum starved cells after exposure to LPS (purple) using the WT-Ovation One-Direct System. The amplified cDNA was labeled and hybridized to Affymetrix GeneChip Human Gene 1.0 ST arrays. Principal Components Analysis (PCA) was performed using Partek Genomics Suite software. An analysis of the array data reveals differential expression of genes consistent with published data on gene expression profiles in LPS stimulation models (not shown).
Highly concordant differential expression results using the Ovation RNA-Seq System on NGS compared with TaqMan data
Differential expression were generated for MAQC A (Human Universal Reference) and MACQ B (Human Brain Reference) by both Illumina Solexa Genome Analyzer IIx and qPCR. RNA-Seq data from 10 ng of total RNA are plotted on X-axis, qPCR plotted on Y-axis. 659 TaqMan probes that uniquely map to the RefSeq annotations used in the RPKM calculations are represented. The qPCR results were originally reported in Shi, L. et al. Nature Biotechnology (2006) 24(9): 1151-1161
Differential expression detected with the Ovation RNA-Seq System on NGS
Data from UCSC genome browser demonstrating differential expression of the TMSB10 gene in Human Brain Reference vs. Universal Human Reference (UHR) MAQC samples. The average #reads/base is 190 for UHR and 69 for Brain Ref, indicating 2.75-fold difference in expression levels.