Comparison of DNA and RNA analyte extraction and melanin removal methods from formalin-fixed, paraffin-embedded (FFPE) melanoma.

• DOI: 10.1200/jco.2015.33.15_suppl.e20002
• Publication Date: 2019-04-19T20:32:07Z
• Authors: Lauren Elaine Haydu; Tiffany L Calderone; John Miller; Roland L. Bassett; Aron Joon; Jianhua Zhang; Margaret B. Morgan; Kenna Rael Shaw; Zachary A Cooper; Elizabeth M Burton; Alan Siroy; Khalida M Wani; Franceso Stingo; Veera Baladandayu...; Michael T. Tetzlaff; Jennifer Ann Wargo; Alexander J. F. L...; Michael A. Davies; Jeffrey E. Gershe...
• Keywords: Molecular diagnostics

e20002 Background: The increasing availability of molecular profiling techniques amenable to FFPE tissue presents a key opportunity study the basis melanoma. In order optimize our methodological approaches for melanoma specimens planned integrative analyses, we compared various DNA and RNA analyte extraction melanin removal methods on yield, quality, profiles. Methods: Primary (n = 2) metastatic 6) with varying degrees pigmentation (3-none/minimal, 3-moderate, 2-high) were used systematic testing 3 4 standard isolation protocols (SIPs). SIPs in combination four potential (Zymo column [Zymo], CTAB, both, none). expression was analyzed using Nanostring 787 custom code set, by next-generation sequencing 202 targeted genes panel [T200]). Results: Significant differences total yield/mg tumor observed between methods. Although all (96/96) analytes passed quality control (QC) regardless SIP, method, or content, gene counts significantly lower 1 SIP. Comparison from remaining 2 revealed no significant counts, hierarchical clustering results, variability above conservative multiplicity-adjusted threshold (Bonferroni, 0.05/770). For isolation, only SIP consistently yielded sufficient highly pigmented specimens. that QC (44 64) run T200 NGS panel. No mutation calls coverage depth. Conclusions: these studies, did not affect Nanostring-based profiling. selected (QiAmp, Zymo) (HighPure, best quantity tumors profiling, provide robust framework ongoing large scale integrated efforts