GU Jiaoyang,NIU Kai,YU Yongxin,et al.Design of a new plasma external reference and analysis of nucleic acid detection[J].J Third Mil Med Univ,2019,41(13):1239-1245.

新型血浆外参照设计及核酸检测分析的研究(/HTML )




Design of a new plasma external reference and analysis of nucleic acid detection
GU Jiaoyang NIU Kai YU Yongxin ZHENG Linpeng YANG Qiao ZHANG Luping SUN Jianguo

Cancer Institute of PLA, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, China

external reference internal reference RT-qPCR miRNA in plasma
R394-33; R394.3; R446.11

目的设计新型血浆外参照(plasma external reference,PLACON),用于人类血浆mRNA、miRNA的检测分析。方法从低级别物种秀丽隐杆线虫基因组中选取一段保守序列,利用BLAST检索PLACON序列的特异性,设计成为新型血浆外参照。以恶性肿瘤患者血浆样品及癌细胞株上清液,利用实时荧光定量PCR法,在血浆或细胞上清液中分别以PLACON、β-actin、GAPDH、U6为参照,检测血浆mRNA、miRNA和细胞上清液mRNA并进行对比分析。结果BLAST检索出PLACON序列具有较好的特异性,与人类基因组无交叉性。熔解曲线均显示RT-qPCR反应特异性较高,PLACON在血浆中mRNA水平扩增的循环阈值(cycle threshold,CT)均显著低于GAPDH、β-actin(P<0.01),在血浆中miRNA水平扩增的CT值显著低于U6(P<0.01)。PLACON在癌细胞上清中进行mRNA扩增实验的验证,得到相似的结果。结论PLACON序列特异性好,在血浆中比内参照更有优势,稳定性更高,扩增效率更高,可用于血浆mRNA、miRNA的定量检测。


ObjectiveTo design a novel plasma external reference (PLACON) to detect and analyze human plasma mRNA and miRNA. MethodsA conserved sequence was selected from the genome of the low-level species Caenorhabditis elegans. It was designed as a novel plasma external reference and BLAST was used to detect the specificity of PLACON sequences. Plasma samples of patients with malignant tumors and cancer cell supernatants were collected. And PLACON, β-actin, GAPDH, and U6 which were added to the plasma or cell supernatant respectively, were used for amplification comparison to detect plasma mRNA, miRNA and cell supernatant mRNA, by quantitative real-time PCR (RT-qPCR). ResultsBLAST detected that the PLACON sequence had good specificity and no cross-correlation with the human genome. Melting curves showed high specificity in RT-qPCR reaction. The cycle threshold values of mRNA levels were significantly lower in plasma of PLACON group than the GAPDH and β-actin groups (P<0.01). The cycle threshold (CT) value of miRNA level amplification in plasma in PLACON group was significantly lower than that in U6 group (P<0.01). PLACON was validated in mRNA amplification experiments in cancer cell supernatants, and similar results were obtained. ConclusionPLACON sequence is specific, and shows better amplification advantage as internal reference in plasma, with high stability and efficient amplification. It can be used for quantitative detection of plasma mRNA and miRNA.


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更新日期/Last Update: 2019-07-08