In recent years, substantial advances have occurred in methodologies for determining gene expression profiles in tissue samples that have been chemically fixed for the purposes of long-term storage. One of the most common techniques for archiving tissue samples has involved formalin fixation, which preserves the cellular RNA but results in substantial degradation during the process of extraction. Thus, to produce enough amplified, labeled product to perform high throughput microarray-based transcriptome profiling, stringent extraction conditions are required in addition to priming amplification of the mRNA both randomly and at the 3’ end.
Because of the wide availability and use of formalin fixed paraffin embedded (FFPE) tissues, several companies have employed considerable effort in developing kits designed specifically for processing these types of samples for microarray analysis. NuGen Technologies, Inc. offers the novel WT-Ovation™ FFPE RNA Amplification System, advertised to perform linear, isothermal, and robust whole transcript amplification from FFPE samples and other sources of degraded RNA. However, unlike other companies that supply kits for amplification of mRNA from FFPE tissue, the WT-Ovation™ amplification technology yields microgram quantities of amplified cDNA instead of cRNA. The amplified product is single-stranded and in the antisense (opposite sense) direction of the mRNA starting material. Because the final product is cDNA, the system is amenable to both downstream microarray and qPCR analysis.
One of the advantages of the WT-Ovation™ FFPE amplification system is its ability to generate amplified cDNA in about 6 hr, as compared to other commercial FFPE amplification kits that typically require 2 rounds of amplification (over 2 days to complete). Starting with 50-100 ng of FFPE-derived total RNA, double-stranded cDNA templates of the mRNA present are synthesized through a reverse transcription reaction. The cDNA template is then amplified linearly by NuGen’s proprietary SPIA™ reagent system. Typical yields range from about 6-10 ug of amplified cDNA and only 5 ug are required for microarray hybridization (in contrast to amplification systems that produce cRNA, where 15 ug of the final product are needed for hybridization). The amplified cDNA is first fragmented and then end-labeled using the FL-Ovation™ Biotin V2 Module prior to overnight hybridization on microarrays.
We have recently began utilizing the WT-Ovation™ FFPE System in our lab to amplify mRNA in laser captured small intestinal epithelial cells and stratified squamous epithelial cells from the oral mucosa for downstream microarray application. These tissues were fixed for 24 to 48 hr in 10% formalin prior to paraffin embedding. About 2000 cells/sample were laser catapulted, in an RNase-free environment, from unstained 6 um tissue sections directly into RNA extraction buffer. 100 ng of total RNA from these extractions was used as the starting material for cDNA synthesis and amplification, yielding about 6-8 ug of final product for fragmentation and labeling. The microarray data generated was in general agreement with our previous expression data from fresh frozen samples of the same tissue.
WT-Ovation™ FFPE System From NuGEN
Rapid; generates cDNA as the final product, allowing both microarray and qPCR based transcriptional assays.
An additional kit must be purchased for the final fragmentation and labeling of the amplified product.
The Bottom Line
A very robust, reproducible linear mRNA amplification system that works reasonably well with formalin fixed samples, which have been traditionally difficult to process for gene expression studies due to inherent degradation of the full length transcripts.