Most clinical tissue samples are preserved using formalin fixation followed by embedding in paraffin. By some estimates, almost 90% of tissue sections are prepared using this methodology. Although this process is excellent for maintaining morphological features, it does not preserve RNA well. In fact, RNA integrity in formalin-fixed, paraffin-embedded (FFPE) tissue is poor. As a result, it is difficult to extract sufficient intact RNA for analysis.
There is much interest in analyzing gene expression in these clinically important samples. For these reasons, there is a strong need to have a reliable and robust approach to extract and prepare for expression analysis the small amount of intact RNA from FFPE-prepared tissues.
Although there are ways to extract RNA from FFPE samples, the degraded character of this RNA makes it difficult to reliably amplify it to produce enough usable material for expression analysis. Most amplification procedures, such as T7 in vitro transcription (T7 IVT), rely on poly-A priming and produce product that is highly biased toward the 3´ end of transcripts. These amplification methods do not typically amplify anything beyond the last 600 to 1,000 bases of the transcript. In addition to leaving much of the RNA inaccessible to analysis, these 3´-biased approaches are particularly problematic with the degraded RNA derived from FFPE tissues, where they typically cannot produce usable product.