Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.
Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.
复制标题
内源性参考基因对基因工程性低芥酸菜籽体事件的数字PCR评估的影响。
DOI：
10.1016/j.bdq.2018.03.002
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发表时间：
2018-05
影响因子：
--
通讯作者：
Eng M
中科院分区：
文献类型：
--
作者：
Demeke T;Eng M
液滴数字PCR（ddPCR）已用于绝对定量遗传工程（GE）事件。通过双重ddPCR绝对定量GE事件需要使用靶基因和参考基因序列的适当引物和探针，以准确测定GE材料的量。单拷贝参考基因通常优选用于通过ddPCR绝对定量GE事件。尚未进行通过ddPCR绝对定量GE卡诺拉事件的参考基因比较研究。研究了四种内源性参考序列（HMG-I/Y、FatA（A）、CruA和Ccf）用于通过ddPCR绝对定量GE卡诺拉事件的适用性。DNA提取方法和DNA质量对参考基因拷贝数评估的影响也进行了研究。ddPCR结果受到单拷贝与双拷贝参考基因的影响。发现单拷贝FatA（A）参考基因是稳定的，并且适用于通过ddPCR绝对定量GE卡诺拉事件。对于所测量的拷贝数，HMG-I/Y参考基因比FatA（A）参考基因更不一致。当使用CruA和Ccf（两个拷贝内源性十字花科植物素序列）时，由于拷贝数较高，预期ddPCR值被低估。如果ddPCR使用两个拷贝的参考基因，为了获得准确的结果，进行调整是很重要的。另一方面，实时定量PCR结果不受使用单拷贝与双拷贝参考基因的影响。
Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences (HMG-I/Y, FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.