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Evaluation and Adaptation of a Laboratory-Based CDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from Up to 35-Year-Old Clinical FFPE Specimens

Abstract

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

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References
1.
Kerlikowske K, Molinaro A, Gauthier M, Berman H, Waldman F, Bennington J . Biomarker expression and risk of subsequent tumors after initial ductal carcinoma in situ diagnosis. J Natl Cancer Inst. 2010; 102(9):627-37. PMC: 2864293. DOI: 10.1093/jnci/djq101. View

2.
Cho W . MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy. Int J Biochem Cell Biol. 2009; 42(8):1273-81. DOI: 10.1016/j.biocel.2009.12.014. View

3.
Moore H, Compton C, Lim M, Vaught J, Christiansen K, Alper J . 2009 Biospecimen research network symposium: advancing cancer research through biospecimen science. Cancer Res. 2009; 69(17):6770-2. PMC: 2782665. DOI: 10.1158/0008-5472.CAN-09-1795. View

4.
Visone R, Croce C . MiRNAs and cancer. Am J Pathol. 2009; 174(4):1131-8. PMC: 2671346. DOI: 10.2353/ajpath.2009.080794. View

5.
Robinson M, McCarthy D, Smyth G . edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009; 26(1):139-40. PMC: 2796818. DOI: 10.1093/bioinformatics/btp616. View