数字PCR定量检测血浆中环状RNA方法的建立
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宁波大学医学院、浙江省病理生理学技术研究重点实验室,宁波 315211

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Tel: 0574-87609589, E-mail: xiaobingxiu@nbu.edu.cnTel: 86-574-87609589, E-mail: xiaobingxiu@nbu.edu.cn

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宁波大学王宽诚基金资助项目.


Establishment of a Method for The Quantitative Detection of Circular RNA in Plasma Using Digital PCR
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Ningbo University School of Medicine, Zhejiang Key Laboratory of Pathophysiology, Ningbo 315211, China

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This work was supported by a grant from the K. C. Wong Magna Fund in Ningbo University.

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    摘要:

    生物标志物一直是医学领域的研究热点. 目前主要以实时荧光定量PCR技术作为分子诊断的检测手段,用于大样本的核酸检测,但该技术具有限制性,会导致假阴性. 作为第三代PCR的数字PCR技术,优化并提高了检测灵敏度,无需标准曲线实现绝对定量检测,大大提高了检测的精准度,尤其是对于低表达RNA的检测,显得更为出色. 本研究以hsa_circ_0061276为例,利用微滴式数字PCR EvaGreen染料法对血浆中hsa_circ_0061276的拷贝数进行绝对定量地检测,成功检测出胃炎和胃癌患者血浆中hsa_circ_0061276的具体拷贝数. 因此,数字PCR作为新型检测技术在临床实际应用中有较大推广价值.

    Abstract:

    Due to the unique structure of circular RNAs (circRNAs) and their widespread presence in body fluids, circRNAs are considered to have the potential as tumor biomarkers. At present, real-time fluorescent quantitative polymerase chain reaction (PCR) technology is thought as one of the main detection methods for molecular diagnosis in clinical practice. However, this technology has limitations with relatively higher false negatives. The digital PCR technology, the third generation PCR, optimizes and improves the detection sensitivity. More important, it does not require a standard curve to achieve absolute quantitative detection, which greatly improving the accuracy of detection, especially for the detection of low-expressed RNAs. In this study, to establish a new method for the quantitative detection of circRNAs in plasma, hsa_circ_0061276 was used as an example. Its specific primers were first designed, and then the economical droplet digital PCR (ddPCR) EvaGreen dye method was used to determine its absolute copy number in plasma samples from 12 patients with gastritis and 13 patients with gastric cancer. Besides, a negative control (without template) was set. The results showed that the total number of droplets in all samples exceeded 14 000. The absolute copy numbers in patients with gastritis are 2.90 Copies/μl, 2.20 Copies/μl, 3.20 Copies/μl, 1.40 Copies/μl, 3.00 Copies/μl, 1.02 Copies/μl, 0.81 Copies/μl, 0.41 Copies/μl, 1.40 Copies/μl, 0.69 Copies/μl, 0.41 Copies/μl, and 2.30 Copies/μl, respectively. The absolute copy numbers in patients with gastric cancer are 1.80 Copies/μl, 2.30 Copies/μl, 0.90 Copies/μl, 0.62 Copies/μl, 0.06 Copies/μl, 3.30 Copies/μl, 0.26 Copies/μl, 0.59 Copies/μl, 1.04 Copies/μl, 0.65 Copies/μl, 0.06 Copies/μl, 1.38 Copies/μl, and 0.18 Copies/μl, respectively. The bands of positive droplets and negative droplets are clearly distinguished, indicating that the specific copy number of hsa_circ_0061276 in plasma has been successfully detected. In conclusion, ddPCR may become a new technology for the detection of DNA/RNA-based biomarkers in biomedical researches and clinical applications with greater promotion value.

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阮垚,郭俊明,肖丙秀.数字PCR定量检测血浆中环状RNA方法的建立[J].生物化学与生物物理进展,2021,48(6):721-726

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  • 收稿日期:2020-12-08
  • 最后修改日期:2021-04-04
  • 接受日期:2021-04-06
  • 在线发布日期: 2021-06-24
  • 出版日期: 2021-06-20
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