Abstract：
Reverse transcription-digital PCR (RT-dPCR) is attracting attention as a method that enables SI-traceable RNA quantification without calibration, but its accuracy and bias have not been thoroughly studied. In this study, the accurate quantification of RNA by the RT-dPCR method was investigated using NMIJ CRM 6204-b, an RNA certified reference material whose certified value was assigned by orthogonal chemical measurement methods. Moreover, a two-step RT-dPCR method was adopted to examine in detail the conditions for the RT reaction process, which was expected to be the major uncertainty component in the RT-dPCR measurement. Optimization experiments revealed that the type of reverse transcriptase, the concentration of template RNA, and the type and concentration of primers in the RT reaction affected the value quantified by RT-dPCR. Under the optimal conditions, the value quantified by RT-dPCR, 76.4 ng/μL ± 6.7 ng/μL (the quantified value ± expanded uncertainty (k = 2)), was consistent with the certified value, 68.2 ng/μL ± 5.8 ng/μL, of NMIJ CRM 6204-b RNA 1000-A within the expanded uncertainty. From the results of the uncertainty evaluation, the relative combined uncertainty of the RT-dPCR method was 4.42%, and the major uncertainty components in the RT-dPCR method were the preparation of RT solution (3.68%), the inter-day difference (1.80%), and the RT reaction (1.30%). Together, the results suggested that the contribution of the RT reaction process to the total uncertainty was greater than that of the dPCR process.
摘要：
逆转录数字PCR（RT-dPCR）作为一种无需校准即可进行SI可追溯RNA定量的方法，引起了人们的注意。但是它的准确性和偏见还没有得到彻底的研究。在这项研究中,使用NMIJCRM6204-b研究了RT-dPCR方法对RNA的准确定量，一种RNA认证的参考材料，其认证值通过正交化学测量方法指定。此外，采用两步RT-dPCR方法详细考察了RT反应过程的条件,这预计将是RT-dPCR测量中的主要不确定度分量。优化实验表明，逆转录酶的类型，模板RNA的浓度，RT反应中引物的类型和浓度影响RT-dPCR定量值。在最优条件下,通过RT-dPCR定量的值，76.4ng/μL±6.7ng/μL（量化值±扩展不确定度（k=2）），与认证值一致，68.2ng/μL±5.8ng/μL，NMIJCRM6204-bRNA1000-A在扩展不确定度范围内。从不确定度评估的结果来看，RT-dPCR方法的相对组合不确定度为4.42%,RT-dPCR方法的主要不确定度分量为RT溶液的制备(3.68%),日间差异(1.80%)，和RT反应(1.30%)。一起,结果表明，RT反应过程对总不确定度的贡献大于dPCR过程。
