Stein Erica V, Duewer David L, Farkas Natalia, Romsos Erica L, Wang Lili, Cole Kenneth D
Biosystems and Biomaterials Division, Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America.
Chemical Sciences Division, Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America.
PLoS One. 2017 Nov 16;12(11):e0188085. doi: 10.1371/journal.pone.0188085. eCollection 2017.
Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.
液滴数字PCR（ddPCR）被倡导作为一种测量罕见基因组靶点的参考方法。一直以来，它在识别DNA拷贝数方面被证明比其他定量方法更灵敏、更直接。然而，使用ddPCR测量微小RNA（miRNA）的最大障碍之一是逆转录效率取决于靶点，这意味着小RNA核苷酸组成直接影响引物特异性，从而阻碍了传统定量优化策略的应用。此外，使用针对定量实时PCR（qRT-PCR）测量miRNA而优化的试剂，在与传统ddPCR定量方法一起使用时，似乎会导致某些靶点出现假阳性或假阴性检测结果。错误读数通常与使用不充分的酶、引物和探针有关。鉴于使用ddPCR进行两步法miRNA定量仅依赖于逆转录，且使用的是以前仅针对qRT-PCR优化的专有试剂，这些障碍是相当大的。因此，在此我们概述了基本的对照、优化技术和一种效能模型以提高ddPCR miRNA测量的质量。我们应用了用于miRNA qRT-PCR测量的两步法原理，并利用合成miRNA靶点来评估使用四种不同商业试剂盒合成cDNA后的ddPCR。我们已经识别出了低效性和局限性，并提出了规避已识别障碍的方法。最后，我们表明我们可以将这些标准应用于一个模型系统，以可靠地定量miRNA拷贝数。我们的测量技术是一种在单个样本中定量特定miRNA拷贝数的新方法，无需为单个实验使用标准曲线。我们的方法可用于验证和对照测量，以及一种诊断技术，使科学家、技术人员、临床医生和监管人员能够基于单一测量单位而非值的比率来进行miRNA测量。
