Virtual Library
Start Your Search
A. Stout
Author of
-
+
P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
-
+
P3.04-025 - DNA Extraction of Lung Cancer Samples for Advanced Diagnostic Testing (ID 3205)
09:30 - 09:30 | Author(s): A. Stout
- Abstract
Background:
Tumor specimens are routinely formalin fixed and paraffin embedded (FFPE) prior to histologic evaluation. This process preserves the morphology and cellular features required for proper staining and microscopic review. However, this practice presents numerous challenges for the extraction of high quality DNA for advanced diagnostic testing that include nanoString and Next-Generation Sequencing (NGS) technologies. An extraction process that consistently produces sufficient DNA yield and fragment size from these difficult but most precious tissue samples is a requirement for any Molecular Pathology laboratory utilizing these platforms. The data presented here will compare the quantity and quality of DNA extracted using two methods, QIAGEN and Covaris, and success of downstream testing.
Methods:
FFPE tumor samples from a variety of tumor types, including lung, were macro-dissected using 14-guage needles, with 1 core extracted using the Covaris truXtract FFPE DNA isolation method and the other matched core using the QIAGEN DNeasy tissue kit. All samples were processed using manufacturer’s recommended instructions. DNA metrics were measured using Qubit (picogreen) and NanoDrop for yield and purity, followed by fragment size estimation on a 2100 BioAnalyzer (Agilent Technologies). A subset of matched DNA sample pairs were used as template for PGM AmpliSeq and MiSeq TSCA library preparation, followed by NGS. A subset of DNA sample pairs were also analyzed for copy number using the nanoString nCounter system.
Results:
DNA yields and fragment lengths were substantially higher for truXtract samples as compared to DNeasy when measured by picogreen quantitation and Bioanalyzer electrophoresis (Figure 1). A higher degree of successful advanced molecular diagnostic test results was also observed for the truXtract DNA samples, especially for the Illumina NGS system (improved clustering and coverage) and nCounter platform (improved counts) that prefer longer fragment lengths than Ion Torrent NGS. Figure 1Figure 1: 2100 Bioanalyzer traces of DNA prepared from three lung cancer FFPE samples; DNeasy (left) and TruXtract (right).
Conclusion:
FFPE tumor samples prepared using the truXtract FFPE DNA isolation kit provides an efficient system for generating high quality DNA samples from even the most difficult lung cancer specimens. The combination of improved yield and fragment size measured for nearly every sample tested suggests that even smaller biopsies can now be collected for advanced diagnostic testing.
