Abstract
BackgroundFormalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA–protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult.MethodsWe excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step.ResultsWe successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well.ConclusionsWe present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.
Highlights
Formalin-fixed paraffin-embedded (FFPE) tissue samples constitute a vast and valuable resource of patient material that can potentially be used for biomedical research
Workflow for the isolation of RNA from normal and cancer‐associated stroma from Formalin-fixed paraffin embedded (FFPE) tissue To isolate normal stroma and cancer-associated stroma (CAS) from within a section of FFPE tissue, we had previously established a workflow for case selection, tissue preparation and staining, Laser-capture microdissection (LCM), and isolation of RNA, which can be followed by RNA analysis by either reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) (Fig. 1) and [8]
The extraction of RNA from FFPE tissues has proved challenging due to chemical crosslinking of RNA with proteins as well as RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. To these difficulties, sample size become very small, e.g. due to the need to use LCM to isolate specific subpopulations of cells, it has been very difficult to recover enough RNA that is amenable for proper analysis using RT-qPCR or NGS. Encountering this problem, we found that commonly used RNA isolation protocols were mostly inefficient due to insufficient initial steps of tissue disruption using proteinase K, which most of the time left behind still macroscopically visible pieces of tissue
Summary
Formalin-fixed paraffin-embedded (FFPE) tissue samples constitute a vast and valuable resource of patient material that can potentially be used for biomedical research. Laser-capture microdissection (LCM) is a technique that allows the specific isolation of defined areas, such as particular subpopulations of cells, from within a tissue section by direct microscopic visualization [5]. This approach enables researchers to precisely dissect areas of interest and address the role(s) of specific subsets of cells within samples of a given pathology directly derived from patients. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult
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