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Comparison between Fluorimetry (Qubit) and Spectrophotometry (NanoDrop) in the Quantification of DNA and RNA Extracted from Frozen and FFPE Tissues from Lung Cancer Patients: A Real-World Use of Genomic Tests.

Author(s): Masago K, Fujita S, Oya Y, Takahashi Y, Matsushita H, Sasaki E, Kuroda H

Publication: Medicina (Kaunas), 2021, Vol. 57, Page

PubMed ID: 34946321 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared quantification of DNA and RNA isolated from surgical, cell block and biopsy lung cancer specimens by spectrophotometry and fluorometry. Additionally, the authors investigated the success rate of genomic testing in specimens with outlier DNA or RNA concentrations. This study included grossly dissected tumor tissue from 585 surgical lung cancer specimens that were snap-frozen in liquid nitrogen, 278 lung cancer biopsy (type unspecified) specimens applied to slides and immersed in 95% ethanol, and 82 cell block specimens (details not provided). Cell block specimens were dewaxed with xylene and treated with proteinase K overnight at 56°C before DNA and RNA extraction.  DNA and RNA were isolated with the QIAamp DNA Mini Kit and RNeasy Mini Kit, respectively. DNA was quantified using the dsDNA HS Assay Kit on a Qubit fluorometer and spectrophotometrically using a NanoDrop machine. The epidermal growth factor receptor (EGFR), KRAS, and BRAF genes were PCR-amplified and sequenced. HER status was analyzed by fragment PCR and anaplastic lymphoma kinase (ALK) was analyzed by immunohistochemistry. AmpliSeq libraries were sequenced on an Ion Torrent S5 Sequencer. The RNA genomic tests were all RT-PCR based.

   Summary of Findings:

    Median purity (A260/A280) of DNA isolated from surgical specimens was slightly higher than from biopsy and cell block specimens (1.88 versus 1.67 and 1.67, respectively) but was comparable between surgical and cell block specimens for RNA (1.98 for surgical, 1.94 for cell block, 1.155 for biopsy). As expected, DNA and RNA concentrations tended to be higher when quantified by spectrophotometer than fluorometry, but there were exceptions. Fluorometric and spectrophotometric quantified DNA concentrations were strongly correlated among cell block specimens (r=0.819, P<0.001), modestly correlated among surgical specimens (r=0.587, P<0.001) and weakly correlated among biopsy specimens (r=0.199, P<0.001). Fluorometric and spectrophotometric quantified RNA concentrations were modestly correlated among cell block  (r=0.597, P<0.001) and surgical specimens (r=0.535, P<0.001) and weakly correlated among biopsy specimens (r=0.181, P<0.001). While the success rate of genomic testing was 96.3% for DNA that had outlier concentrations by Qubit, the success rate for specimens with an outlier RNA concentration ranged from 39.3% (ALK-Fusion) to 60.7% for KRAS.  DNA and RNA NGS analysis of specimens that had an outlier concentration determined by Qubit were successful in 77.8% (14/18) and 91.5% (43/47), respectively

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin
   • Ethanol
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Spectrophotometry
   DNA	PCR
   DNA	Next generation sequencing
   Protein	Immunohistochemistry
   RNA	Spectrophotometry
   RNA	RT-PCR
   RNA	Next generation sequencing
   RNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Spectrophotometry Specific	Technology platform	Spectrophotometry Qubit dsDNA HS Assay Kit
   Biospecimen Acquisition	Method of tissue acquisition	Biopsy Surgical resection

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