Feasibility of RNA and DNA Extraction from Fresh Pipelle and Archival Endometrial Tissues for Use in Gene Expression and SNP Arrays

Abstract

Identifying molecular markers of endometrial hyperplasia (neoplasia) progression is critical to cancer prevention. To assess RNA and DNA quantity and quality from routinely collected endometrial samples and evaluate the performance of RNA- and DNA-based arrays across endometrial tissue types, we collected fresh frozen (FF) Pipelle, FF curettage, and formalin-fixed paraffin-embedded (FFPE) hysterectomy specimens (benign indications) from eight women. Additionally, neoplastic and uninvolved tissues from 24 FFPE archival hysterectomy specimens with endometrial hyperplasias and carcinomas were assessed. RNA was extracted from 15 of 16 FF and 51 of 51 FFPE samples, with yields >1.2 μg for 13/15 (87%) FF and 50/51 (98%) FFPE samples. Extracted RNA was of high quality; all samples performed successfully on the Illumina whole-genome cDNA-mediated annealing, selection, extension, and ligation (WG-DASL) array and performance did not vary by tissue type. While DNA quantity from FFPE samples was excellent, quality was not sufficient for successful performance on the Affymetrix SNP Array 6.0. In conclusion, FF Pipelle samples, which are minimally invasive, yielded excellent quantity and quality of RNA for gene expression arrays (similar to FF curettage) and should be considered for use in genomic studies. FFPE-derived DNA should be evaluated on new rapidly evolving sequencing platforms.

Genetic Signatures From RNA Sequencing of Pediatric Localized Scleroderma Skin

“…Traditionally, NGS techniques including RNAseq have employed fresh or fresh-frozen (FF) samples rather than formalin-fixed, paraffin-embedded (FFPE) samples due to the potential for degradation, with decreased exonic reads and increased intronic reads with FFPE samples (13). However, more recent studies of other human tissue, including brain, endometrial, lung, and breast cancers (14)(15)(16)(17)(18)(19)(20)(21)(22), demonstrated that FFPE samples stored for up to 32 years have been successfully analyzed with RNAseq, introducing the potential for future NGS analysis of widely available archival FFPE samples (23). FFPE samples are more readily available for children who have undergone a diagnostic biopsy, and eliminate the need for a separate research procedure.…”

Genomic, transcriptomic, and viral integration profiles associated with recurrent/metastatic progression in high‐risk human papillomavirus cervical carcinomas

“…First, our study had a small sample size precluding definite conclusions which normally require a larger sample size to ensure a representative distribution of the population and to be considered representative of groups of people by statistical tests. Second, the use of FFPE tissue archives instead of fresh‐frozen (FF) tissues may be associated with known artifacts with impacts on the sequencing and the fidelity of expression profiles as previously proposed with limited quantity of DNA from FFPE samples 69‐71 . Lastly, the validation of targeted RNA‐seq by reverse transcriptase polymerase chain reaction (RT‐PCR) and HPV integration sites by fluorescent in situ hybridization (FISH) or PCR should be considered in the further study.…”

“…Second, the use of FFPE tissue archives instead of fresh-frozen (FF) tissues may be associated with known artifacts with impacts on the sequencing and the fidelity of expression profiles as previously proposed with limited quantity of DNA from FFPE samples. [69][70][71] Lastly, the validation of targeted RNA-seq by reverse transcriptase polymerase chain reaction (RT-PCR) and HPV integration sites by fluorescent in situ hybridization (FISH) or PCR should be considered in the further study.…”

Endometrial Carcinoma, Grossing and Processing Issues: Recommendations of the International Society of Gynecologic Pathologists

“…Procurement of fresh tissue for banking or research protocols should be done as soon as possible as prolonged ischemia affects tissue quality for investigational purposes. Tissue procurement for research should not compromise pathologic evaluation 6–10. Therefore, a pathologist should be consulted in questionable cases.…”