Citation: Yang Dandan, Sun Yuanyuan, Chang Fu, Tian Hui, Liu Chenghui, Li Zhengping. Highly specific quantification of mRNA mutation in single cells based on RNase H cleavage-assisted reverse transcription (RT)-PCR[J]. Chinese Chemical Letters, ;2020, 31(5): 1095-1098. doi: 10.1016/j.cclet.2019.09.015 shu

Highly specific quantification of mRNA mutation in single cells based on RNase H cleavage-assisted reverse transcription (RT)-PCR

Yang Dandan ^a,1 ,
Sun Yuanyuan ^b,1 ,
Chang Fu ^a ,
Tian Hui ^a ,
Liu Chenghui ^a,*, ,
Li Zhengping ^a,*,

a.
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China
b.
The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

liuch@snnu.edu.cn

lzpbd@snnu.edu.cn

Received Date: 12 August 2019
Revised Date: 6 September 2019
Accepted Date: 9 September 2019
Available Online: 10 September 2019

Figures(4)

Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies. How to eliminate the false-positive interference from the abundant normal mRNA is still a big challenge. Herein, we have proposed an LNA (locked nucleic acid)-assisted high-specificity strategy which can selectively guide the RNase H to cleave only the wildtype mRNA (wtRNA) while the mutant mRNA (mutRNA) will remain intact. The intact mutRNA can be amplified and detected by real-time reverse transcription (RT)-PCR but the disconnected wtRNA will be not replicated at all. Based on the highly selective depletion of wtRNA, this elegant design effectively avoids the false-positive interference from the high background of normal mRNA and thus can guarantee the accurate and reliable detection of rare mutRNA in real biomedical samples. Besides for the excellent specificity, ultrahigh sensitivity is also achieved for this proposed assay, which allows the quantification of mutRNA at single molecule and single cell level. Due to its easy design, high sensitivity and specificity, the established LNA probe-assisted RT-PCR strategy provides a powerful tool for studying the function of mutRNA at the single cell level and for the mutRNA-associated liquid biopsy.
- mRNA mutation,
- Single cell,
- LNA probe,
- RNase H,
- RT-PCR
1. [1]
  M.G. Mohsen, D.B. Ji, E.T. Kool, Chem. Sci. 10 (2019) 3264-3270. doi: 10.1039/C8SC03901A
2. [2]
  S. Levy, G. Sutton, P.C. Ng, et al., PLoS Biol. 5 (2007) e254. doi: 10.1371/journal.pbio.0050254
3. [3]
  J.B. Long, Y.X. Liu, Q.F. Cao, et al., Chin. Chem. Lett. 26 (2015) 1031-1035. doi: 10.1016/j.cclet.2015.05.036
4. [4]
  D.B. Zhu, D. Xing, Y.B. Tang, Chin. Chem. Lett. 18 (2007) 869-871. doi: 10.1016/j.cclet.2007.05.002
5. [5]
  J. Das, I. Ivanov, L. Montermini, et al., Nat. Chem. 7 (2015) 569-575. doi: 10.1038/nchem.2270
6. [6]
  C.E. Meacham, S.J. Morrison, Nature 501 (2013) 328-337. doi: 10.1038/nature12624
7. [7]
  C.H. Zong, S.J. Lu, A.R. Chapman, et al., Science 338 (2012) 1622-1626. doi: 10.1126/science.1229164
8. [8]
  A. Rakszewska, R.J. Stolper, A.B. Kolasa, et al., Angew. Chem. Int. Ed. 55 (2016) 6698-6701. doi: 10.1002/anie.201601969
9. [9]
  C. Larsson, I. Grundberg, O. Söderberg, et al., Nat. Methods 7 (2010) 395-397. doi: 10.1038/nmeth.1448
10. [10]
  S.Z. Yue, X.Y. Song, W.L. Song, et al., Chem. Sci. 10 (2019) 1651-1658. doi: 10.1039/C8SC04756A
11. [11]
  Y.F. Li, S.Z. Yue, H.J. Qi, et al., Chem. Commun. 55 (2019) 4103-4106. doi: 10.1039/C9CC00747D
12. [12]
  S. Bi, S.Z. Yue, S.S. Zhang, Chem. Soc. Rev. 46 (2017) 4281-4298. doi: 10.1039/C7CS00055C
13. [13]
  X.D. Li, B. Chen, L. Lan, et al., Chin. Chem. Lett. 29 (2018) 1637-1640. doi: 10.1016/j.cclet.2018.06.003
14. [14]
  C.B. Qi, H.P. Jiang, J. Xiong, et al., Chin. Chem. Lett. 30 (2019) 553-557. doi: 10.1016/j.cclet.2018.11.029
15. [15]
  J. Morlan, J. Baker, D. Sinicropi, PLoS One 4 (2009) e4584. doi: 10.1371/journal.pone.0004584
16. [16]
  G.M. Arechavaleta, V. Scholl, V. Pérez, et al., Clin. Exp. Med. 11 (2011) 55-59. doi: 10.1007/s10238-010-0101-x
17. [17]
  W.W. Chen, L. Balaj, L.M. Liau, et al., Mol. Ther.-Nucl. Acids 2 (2013) e109. doi: 10.1038/mtna.2013.28
18. [18]
  Y.L. Hu, H.X. Jia, Y.C. Wang, et al., Chem. Commun. 50 (2014) 13093-13095. doi: 10.1039/C4CC05102E
19. [19]
  D.J. Panka, R.J. Sullivan, J.W. Mier, Melanoma Res. 20 (2010) 401-407.
20. [20]
  D. Dankort, D.P. Curley, R.A. Cartlidge, et al., Nat. Genet. 41 (2009) 544-552. doi: 10.1038/ng.356
21. [21]
  Y. You, B.G. Moreira, M.A. Behlke, et al., Nucleic Acids Res. 34 (2006) e60. doi: 10.1093/nar/gkl175
22. [22]
  C.Y. Lee, H. Jang, K.S. Park, et al., Nanoscale 9 (2017) 16149-16153. doi: 10.1039/C7NR04060A
23. [23]
  M. Nowotny, S.A. Gaidamakov, R.J. Crouch, et al., Cell 121 (2005) 1005-1016. doi: 10.1016/j.cell.2005.04.024
24. [24]
  B. Sapkota, C.E. Hill, B.P. Pollack, OncoImmunology 2 (2013) e22890. doi: 10.4161/onci.22890
25. [25]
  A. Nakamura, T. Arita, S. Tsuchiya, et al., Cancer Res. 73 (2013) 7043-7055. doi: 10.1158/0008-5472.CAN-13-1825
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