Nakayama Yuki, Yamaguchi Hiromi, Einaga Naoki, Esumi Mariko
Department of Pathology, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan.
PLoS One. 2016 Mar 3;11(3):e0150528. doi: 10.1371/journal.pone.0150528. eCollection 2016.
The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR.
荧光定量仪是一种基于荧光染料与双链DNA（dsDNA）结合的荧光强度进行DNA定量的设备。由于它能测量完整的双链DNA，荧光定量仪通常被认为对于下一代测序前检查DNA质量很有用。为了研究用于质量评估的最准确和合适的DNA定量方法，我们比较了三种定量方法：测量紫外吸光度的NanoDrop、荧光定量仪以及测量目标基因丰度的定量PCR（qPCR）。为了进行比较，我们使用了三种类型的DNA：1）从新鲜冷冻肝组织中提取的DNA（冷冻DNA）；2）从与冷冻DNA所用组织相当的福尔马林固定、石蜡包埋肝组织中提取的DNA（FFPE-DNA）；3）用Trizol试剂提取RNA后从剩余部分中提取的DNA（Trizol-DNA）。这些DNA用蒸馏水进行系列稀释，并用三种定量方法进行测量。对于冷冻DNA，与NanoDrop和qPCR值相反，荧光定量仪的值与稀释倍数不成比例。荧光定量仪值的这种非比例下降取决于较低的盐浓度，并且DNA溶液中NaCl浓度超过1 mM才能进行荧光定量仪测量。对于FFPE-DNA，荧光定量仪的值与稀释倍数成比例，并且低于NanoDrop值。然而，电泳显示qPCR比荧光定量仪更准确地反映了DNA片段化程度。因此，在检查FFPE-DNA质量方面，qPCR优于荧光定量仪。对于Trizol-DNA，荧光定量仪的值与稀释倍数成比例，并且始终低于NanoDrop值，与FFPE-DNA类似。然而，qPCR值高于NanoDrop值。用SYBR Green I进行的电泳和单链DNA（ssDNA）定量表明，Trizol-DNA主要由未片段化的ssDNA组成。因此，荧光定量仪并不总是定量可用于PCR的DNA的最准确方法。
