Detection of plasmatic trace amounts of microRNAs with two kinds of small RNA real-time polymerase chain reaction methods_West China Medical Journal

Authors：

CHEN Xuemei , DING Yu ,  WU Sisi

Core Facility of West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding?author：

WU Sisi, Email: 949189109@qq.com

Keywords：

MicroRNAs; Stem-loop; Poly A polymerase; Real-time quantitative polymerase chain reaction; Plasma

DOI：

10.7507/1002-0179.201604236

Video：

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Objective To detect traces of microRNAs (miRNAs) in plasma and assess the expression stability of two common reference genes by stem-loop and poly A polymerase (PAP) real-time quantitative polymerase chain reaction (PCR) method, as miRNAs are the new bio-markers of tumor diagnosis and molecular targeted therapy, and its quantitative research is very important. Methods We extracted miRNAs from plasma of adult Sprague Dawley (SD) rats’ plasma, and detected the expressions of rno-miR-200b-3p and rno-miR-126-3p with stem-loop and PAP real-time PCR quantitative method, with rno-miR-103a-3p and U6 as internal controls. All the results were evaluated by 2^△Ct method. Results Compared with PAP method, the stem-loop method reduced Ct value by 2-4 cycles and improved sensitivity by 10 times. In PAP method, the melting curve showed two peaks, a main peak and a small non-specific peak. Yet the melting curve of stem-loop method demonstrated a single specific peak. Furthermore, we validated the stability of internal references in the two real time PCR methods. U6 presented a more stable Ct value than rno-miR-103 in adult SD rats’ plasma samples. Conclusions Stem-loop real-time PCR is recommended as a major way to detect some samples with a low concentration of miRNAs, owing to its high accuracy and sensitivity. However, if a large number of tissue samples is going to be detected, PAP real-time PCR is more suitable and convenient than stem-loop method. U6 is more stable and repeatable than rno-miR-103a-3p as the reference gene to evaluate the semi-quantitative consequence of miRNAs.

Citation： CHEN Xuemei, DING Yu, WU Sisi. Detection of plasmatic trace amounts of microRNAs with two kinds of small RNA real-time polymerase chain reaction methods. West China Medical Journal, 2017, 32(5): 699-704. doi: 10.7507/1002-0179.201604236 Copy

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11.	吳瑞珊, 溫旺榮. 外周血 miRNA 的診斷意義. 分子診斷與治療雜志, 2012, 4(2): 131-136.
12.	宋嘉哲, 孫福伯, 莊開歌, 等. 基于實時熒光定量反轉錄 PCR 技術的動物 miRNA 表達分析策略. 中國畜牧獸醫, 2013, 40(9): 109-113.
13.	陳旭. miRNA 定量檢測中內參基因的選擇. 國際檢驗醫學雜志, 2012, 33(11): 1338-1340.
14.	Osaki M, Takeshita F, Ochiya T. MicroRNAs as biomarkers and therapeutic drugs in human cancer. Biomarkers, 2008, 13(7): 658-670.
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17.	張曉娟, 董靜, 馬紅霞. 血清/血漿 microRNA 檢測方法探討與建立. 南京醫科大學學報: 自然科學版, 2011, 31(4): 529-531.
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19.	Wu XM, Liu MY, Ge XX, et al. Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange. Planta, 2011, 233(3): 495-505.

1. ?Tang G. siRNA and miRNA: an insight into RISCs. Trends Biochem Sci, 2005, 30(2): 106-114.
2. Buckingham M. Myogenic progenitor cells and skeletal myogenesis in vertebrates. Curr Opin Genet Dev, 2006, 16(5): 525-532.
3. 時宿妹, 張越, 張傳宇, 等. 2 種微小 RNA 實時定量 PCR 檢測方法的比較. 生物技術通訊, 2010, 21(3): 377-384.
4. 趙麗, 楊洋, 溫傳俊. 莖-環 RT-PCR 法定量 miRNA-421 的引物設計. 南京師大學報: 自然科學版, 2012, 35(2): 83-88.
5. 張宇, 張勇, 楊長成, 等. microRNA 表達檢測技術進展. 新鄉醫學院學報, 2008, 25(6): 634-637.
6. 丁琦晨, 鄭敏. 血液 miRNA 與肺癌關系的研究進展. 中國臨床醫學, 2015, 22(4): 568-570.
7. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA, 2008, 105(30): 10513-10518.
8. 楊東, 張紅. MicroRNAs-126(miR-126)的生物學功能. 現代生物醫學進展, 2012, 12(14): 2773-2777.
9. 杜葉平, 吳潔, 武春梅, 等. miR-200b 在結直腸癌組織中表達情況及臨床意義. 國際檢驗醫學雜志, 2013, 34(16): 2095-2098.
10. 陳志凱, 傅松維, 鮑舟君, 等. 胃癌患者血清 miR-200b 和 miR-200c 水平變化及臨床意義. 中國生化藥物雜志, 2016, 5(5): 201-203, 206.
11. 吳瑞珊, 溫旺榮. 外周血 miRNA 的診斷意義. 分子診斷與治療雜志, 2012, 4(2): 131-136.
12. 宋嘉哲, 孫福伯, 莊開歌, 等. 基于實時熒光定量反轉錄 PCR 技術的動物 miRNA 表達分析策略. 中國畜牧獸醫, 2013, 40(9): 109-113.
13. 陳旭. miRNA 定量檢測中內參基因的選擇. 國際檢驗醫學雜志, 2012, 33(11): 1338-1340.
14. Osaki M, Takeshita F, Ochiya T. MicroRNAs as biomarkers and therapeutic drugs in human cancer. Biomarkers, 2008, 13(7): 658-670.
15. Gilad S, Meiri E, Yogev Y, et al. Serum microRNAs are promising novel biomarkers. PLoS One, 2008, 3(9): e3148.
16. Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res, 2008, 18(10): 997-1006.
17. 張曉娟, 董靜, 馬紅霞. 血清/血漿 microRNA 檢測方法探討與建立. 南京醫科大學學報: 自然科學版, 2011, 31(4): 529-531.
18. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res, 2005, 33(20): e179.
19. Wu XM, Liu MY, Ge XX, et al. Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange. Planta, 2011, 233(3): 495-505.

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West China Medical Journal

Detection of plasmatic trace amounts of microRNAs with two kinds of small RNA real-time polymerase chain reaction methods

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