NDLI: Results and lessons from dual extraction of DNA and RNA from formalin-fixed paraffin-embedded breast tumor tissues for a large Cancer epidemiologic study

Content Provider	Springer Nature : BioMed Central
Author	Ondracek, Rochelle Payne Chen, Jianhong Marosy, Beth Szewczyk, Sirinapa Medico, Leonard Mohan, Amrutha Sherly Nair, Priya Pratt, Rachel Roh, Janise M. Khoury, Thaer Carpten, John Kushi, Lawrence H. Palmer, Julie R. Doheny, Kim Davis, Warren Higgins, Michael J. Yao, Song Ambrosone, Christine B.
Abstract	Background The use of archived formalin-fixed paraffin-embedded (FFPE) tumor tissues has become a common practice in clinical and epidemiologic genetic research. Simultaneous extraction of DNA and RNA from FFPE tissues is appealing but can be practically challenging. Here we report our results and lessons learned from processing FFPE breast tumor tissues for a large epidemiologic study. Methods Qiagen AllPrep DNA/RNA FFPE kit was adapted for dual extraction using tissue punches or sections from breast tumor tissues. The yield was quantified using Qubit and fragmentation analysis by Agilent Bioanalyzer. A subset of the DNA samples were used for genome-wide DNA methylation assays and RNA samples for sequencing. The QC metrices and performance of the assays were analyzed with pre-analytical variables. Results A total of 1859 FFPE breast tumor tissues were processed. We found it critical to adjust proteinase K digestion time based on tissue volume to achieve balanced yields of DNA and RNA. Tissue punches taken from tumor-enriched regions provided the most reliable output. A median of 1475 ng DNA and 1786 ng RNA per sample was generated. The median DNA integrity number (DIN) was 3.8 and median DV200 for RNA was 33.2. Of 1294 DNA samples used in DNA methylation assays, 97% passed quality check by qPCR and 92% generated data deemed high quality. Of the 130 RNA samples with DV200 ≥ 20% used in RNA-sequencing, all but 5 generated usable transcriptomic data with a mapping rate ≥ 60%. Conclusions Dual DNA/RNA purification using Qiagen AllPrep FFPE extraction protocol is feasible for clinical and epidemiologic studies. We recommend tissue punches as a reliable source material and fine tuning of proteinase K digestion time based on tissue volume. Impact Our protocol and recommendations may be adapted by future studies for successful extraction of archived tumor tissues.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-022-08837-6.pdf
Ending Page	10
Page Count	10
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/s12864-022-08837-6
Journal	BMC Genomics
Issue Number	1
Volume Number	23
Language	English
Publisher	BioMed Central
Publisher Date	2022-08-25
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics FFPE Population study Nucleic acids extraction Breast tumor
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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