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.
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.
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DOI：
10.3390/medicina57121375
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发表时间：
2021-12-17
期刊：
影响因子：
--
通讯作者：
Kuroda H
中科院分区：
文献类型：
--
作者：
Masago K;Fujita S;Oya Y;Takahashi Y;Matsushita H;Sasaki E;Kuroda H
背景和目的：基于面板的下一代测序（NGS）已在日常临床环境中进行，用于非小细胞肺癌（NSCLC）患者的诊断和治疗指导。包括NGS在内的基因组检测的成功在很大程度上取决于制备质量更好的DNA或RNA;然而，目前还没有建立用于制备基因组DNA和RNA样品的操作方法。材料与方法：我们比较了以下两种定量方法，QubitTM和NanoDropTM，使用585例手术标本，278例活检标本和82例用于基因检测的肺癌细胞块标本，包括NGS。我们分析了基因组测试的成功率，包括NGS，这些测试是用DNA和RNA进行的，浓度是Qubit荧光计的离群值。结果如下：使用NanoDropTM测量时，无论样本类型如何，DNA浓度的绝对值都有较高的趋势;然而，RNA的情况并非如此。使用浓度低于QubitTM检测下限的样本进行DNA基因组检测的成功率高达约96%。基于RNA的基因组检测（包括RT-PCR）的成功率低于60%，并不令人满意。AmpliSeqTM DNA panel测序和RNA panel测序的成功率分别为77.8%和91.5%。如果可以获得至少一个PCR扩增产物，则成功地进行了所有基于RNA的测序。结论：NanoDropTM浓度测量是可靠的。浓度低于QubitTM检测限的样本的NGS成功率相对高于预期，值得进行基于PCR的面板测序，特别是在无法进行重新活检的情况下。
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.
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影响因子：
6.4
作者：
Sedlackova T;Repiska G;Celec P;Szemes T;Minarik G
通讯作者：
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