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

• Published in: Medicina (Kaunas, Lithuania) Vol. 57; no. 12; p. 1375
• Main Authors: Masago, Katsuhiro, Fujita, Shiro, Oya, Yuko, Takahashi, Yusuke, Matsushita, Hirokazu, Sasaki, Eiichi, Kuroda, Hiroaki
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 17.12.2021; MDPI
• Subjects: Biopsy; Carcinoma, Non-Small-Cell Lung - genetics; DNA; Fluorometry; Genomics; Humans; Lung cancer; Lung Neoplasms - genetics; Methods; Mutation; next-generation sequencing; PCR-based; Regression analysis; RNA - genetics; Spectrophotometry; Statistical analysis; Success; Surgery; UV absorbance measurement; United States > US; PCR-based; next-generation sequencing; UV absorbance measurement
• ISSN: 1648-9144; 1010-660X; 1648-9144
• DOI: 10.3390/medicina57121375

Panel-based next-generation sequencing (NGS) has been carried out in daily clinical settings for the diagnosis and treatment guidance of patients with non-small cell lung cancer (NSCLC). The success of genomic tests including NGS depends in large part on preparing better-quality DNA or RNA; however, there are no established operating methods for preparing genomic DNA and RNA samples. We compared the following two quantitative methods, the QubitTM and NanoDropTM, using 585 surgical specimens, 278 biopsy specimens, and 82 cell block specimens of lung cancer that were used for genetic tests, including NGS. We analyzed the success rate of the genomic tests, including NGS, which were performed with DNA and RNA with concentrations that were outliers for the Qubit Fluorometer. The absolute value for DNA concentrations had a tendency to be higher when measured with NanoDropTM regardless of the type of specimen; however, this was not the case for RNA. The success rate of DNA-based genomic tests using specimens with a concentration below the lower limit of QubitTM detection was as high as approximately 96%. At less than 60%, the success rate of RNA-based genomic tests, including RT-PCR, was not as satisfactory. The success rates of the AmpliSeqTM DNA panel sequencing and RNA panel sequencing were 77.8% and 91.5%, respectively. If at least one PCR amplification product could be obtained, then all RNA-based sequencing was performed successfully. The concentration measurements with NanoDropTM are reliable. The success rate of NGS with samples at concentrations below the limit of detection of QubitTM was relatively higher than expected, and it is worth performing PCR-based panel sequencing, especially in cases where re-biopsy cannot be performed.