Results and lessons from dual extraction of DNA and RNA from formalin-fixed paraffin-embedded breast tumor tissues for a large Cancer epidemiologic study.
Breast tumor
FFPE
Nucleic acids extraction
Population study
Journal
BMC genomics
ISSN: 1471-2164
Titre abrégé: BMC Genomics
Pays: England
ID NLM: 100965258
Informations de publication
Date de publication:
25 Aug 2022
25 Aug 2022
Historique:
received:
12
04
2022
accepted:
03
08
2022
entrez:
25
8
2022
pubmed:
26
8
2022
medline:
30
8
2022
Statut:
epublish
Résumé
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. 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. 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%. 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. Our protocol and recommendations may be adapted by future studies for successful extraction of archived tumor tissues.
Sections du résumé
BACKGROUND
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
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
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
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
CONCLUSIONS
Our protocol and recommendations may be adapted by future studies for successful extraction of archived tumor tissues.
Identifiants
pubmed: 36008758
doi: 10.1186/s12864-022-08837-6
pii: 10.1186/s12864-022-08837-6
pmc: PMC9404650
doi:
Substances chimiques
Formaldehyde
1HG84L3525
RNA
63231-63-0
DNA
9007-49-2
Endopeptidase K
EC 3.4.21.64
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
614Subventions
Organisme : NCI NIH HHS
ID : U01 CA195565
Pays : United States
Organisme : NCI NIH HHS
ID : U01 CA164974
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA225947
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA105274
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA247281
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA228156
Pays : United States
Organisme : NCI NIH HHS
ID : U54 CA233444
Pays : United States
Informations de copyright
© 2022. The Author(s).
Références
J Oncol. 2009;2009:871250
pubmed: 19865486
Cancer Causes Control. 2014 Mar;25(3):309-19
pubmed: 24343304
Anal Biochem. 2010 May 1;400(1):110-7
pubmed: 20079706
BMC Cancer. 2016 Aug 30;16:692
pubmed: 27578032
NPJ Breast Cancer. 2020 Sep 21;6:45
pubmed: 33024820
PLoS One. 2014 May 30;9(5):e98187
pubmed: 24878701
PLoS One. 2014 Aug 08;9(8):e104566
pubmed: 25105902
BMC Genomics. 2015 Nov 03;16:892
pubmed: 26531060
PLoS One. 2018 Apr 5;13(4):e0195471
pubmed: 29621323
Exp Ther Med. 2017 Sep;14(3):2683-2688
pubmed: 28962212
Cancer Causes Control. 2008 Dec;19(10):1065-76
pubmed: 18478338
PLoS One. 2017 Jun 22;12(6):e0179732
pubmed: 28640876
PLoS One. 2015 Dec 07;10(12):e0144162
pubmed: 26641479
Virchows Arch. 2012 Feb;460(2):131-40
pubmed: 22270699