In a groundbreaking study, researchers from the National Cancer Institute have uncovered critical insights into the effects of formalin fixation and delay to fixation on RNA and miRNA-Seq profiles
Formalin-fixation and delays to fixation (DTF) are common procedures in tissue preservation, particularly in the context of cancer diagnostics. However, the ramifications of these processes on molecular analyses have been a matter of ongoing investigation. The National Cancer Institute’s recent study leverages Next-Generation Sequencing (NGS) technology to elucidate the effects of these pre-analytical variables on RNA integrity and miRNA expression. Findings suggest that while DTF has negligible effects on NGS analysis, formalin-induced changes significantly alter the relative proportions of intronic, exonic, and untranslated RNA segments captured by RNA-seq.
DOI: (https://doi.org/10.1038/s41598-019-43282-8)
The study, published in the journal Scientific Reports under the title “Deleterious effects of formalin-fixation and delays to fixation on RNA and miRNA-Seq profiles” (Sci Rep 9, 6980), details the comparison of whole transcriptome sequencing and small RNA profiling of matched snap-frozen and Formalin-Fixed Paraffin-Embedded (FFPE) specimens. The goal was to establish acceptable DTFs and optimal workflow for reliable FFPE specimen analysis via NGS.
Study Design and Findings
Under the aegis of the Biospecimen Pre-analytical Variables (BPV) study, researchers employed a comprehensive approach by sequencing RNA from matched snap-frozen and FFPE samples subjected to varying DTFs. Notably, evidence from the project points to formalin fixation distorting the representation of RNA regions for most genes compared to snap-freezing. Conversely, DTF presented minimal influence on the NGS results.
A crucial revelation of the research indicated that 80% of specimens showcased a set of RNAs that consistently differed between snap-frozen and FFPE specimens across tissue types. Surprisingly, this RNA subset remained consistent in the remaining 20% of specimens. This finding suggests a formalin-induced signature on RNA that may be independent of tissue type.
Contrastingly, miRNA expression displayed a different picture. While generally stable across various formalin fixation protocols, a 12-hour DTF induced increased variability in miRNA expression. This variability underlines the imperative for timely processing of formalin-fixed samples to preserve miRNA integrity for subsequent sequencing.
As for the implications, this research underscores the importance of standardized tissue handling protocols in clinical and research settings. The integrity of nucleic acids in FFPE samples is critical for accurate diagnostic and research outcomes, particularly as molecular profiling becomes integral to precision medicine.
Significance and Future Directions
The insights derived from the BPV study are valuable for refining practices regarding biospecimen handling, which is vital for the future of cancer research and diagnostics. Researchers and clinicians can leverage these findings to ensure that the nucleic acids extracted from FFPE samples, which are key in identifying genetic drivers of cancer and therapeutic targets, maintain their integrity for analysis.
Moreover, this research propels a discussion regarding the development of advanced fixatives and preservation methods that could minimize genetic and molecular distortions otherwise introduced by formalin. Continual improvement in tissue preservation and processing may further enhance the fidelity of molecular analyses conducted on FFPE samples.
References
1. Jones, W. W., et al. (2019). Deleterious effects of formalin-fixation and delays to fixation on RNA and miRNA-Seq profiles. Scientific Reports, 9, 6980. doi: (https://doi.org/10.1038/s41598-019-43282-8)
2. Xuan, J., et al. (2013). Next-generation sequencing in the clinic: promises and challenges. Cancer Letters, 340, 284-295. doi:(https://doi.org/10.1016/j.canlet.2012.11.025)
3. Carrick, D. M., et al. (2015). Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue. PLoS One, 10, e0127353. doi:(https://doi.org/10.1371/journal.pone.0127353)
4. Lesluyes, T., et al. (2016). RNA sequencing validation of the Complexity INdex in SARComas prognostic signature. European Journal of Cancer, 57, 104–111. doi:(https://doi.org/10.1016/j.ejca.2015.12.027)
5. Li, P., et al. (2014). Whole-Transcriptome profiling of formalin-fixed, paraffin-embedded renal cell carcinoma by RNA-seq. BMC Genomics, 15, 1087. doi:(https://doi.org/10.1186/1471-2164-15-1087)
Keywords
1. RNA integrity in FFPE samples
2. FFPE NGS analysis reliability
3. Formalin fixation RNA effects
4. Delay to fixation miRNA variability
5. Standardized biospecimen handling protocols
Overall, the exhaustive analysis of RNA and miRNA under varying conditions of fixation and handling has paved the way for a more robust understanding of pre-analytical variables. Such knowledge proves essential for enhancing the quality of cancer research and patient care in an era increasingly defined by detailed genomic landscapes.