Abstract
Background and Objectives: 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. Materials and Methods: 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. Results: 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. Conclusions: 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.
Highlights
Preparing better quality DNA or RNA is more dependent on other pre-analytic procedures, such as what happens after the surgery, fixation period, fixation procedure in general, procedures in a pathology laboratory, and nucleic acid extraction protocols, which are included in the Standard PRE analytical Code (SPREC) [6]; the accuracy of the measurement of DNA or RNA has become more important
The absolute value of DNA concentration had a tendency to be higher when measured with NanoDrop regardless of the type of specimen
At less than 60%, the success rate of the RNA-based genomic test, cluding RT-PCR, was not as satisfactory
Summary
Somatic mutations of EGFR and BRAF and gene rearrangements of ALK and ROS1 have been recommended for testing before the initial treatment of patients with advanced non-small cell lung cancer (NSCLC) based on the guidelines from major professional organizations [1,2,3,4,5]. Preparing better quality DNA or RNA is more dependent on other pre-analytic procedures, such as what happens after the surgery, fixation period, fixation procedure in general (especially for the majority of the labs which work with formalin fixed paraffin embedded tissues), procedures in a pathology laboratory, and nucleic acid extraction protocols, which are included in the Standard PRE analytical Code (SPREC) [6]; the accuracy of the measurement of DNA or RNA has become more important. The preparation of a proper DNA or RNA extraction procedure and the establishment of an accurate quantification method of these samples play a key role in the success of these genomic tests; there are no established operating methods for preparing genomic DNA and RNA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
