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Fluorometric determination of microRNA based on strand displacement amplification and rolling circle amplification

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Abstract

The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.

Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.

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References

  1. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843–854. doi:10.1016/0092-8674(93)90529-Y

    Article  CAS  Google Scholar 

  2. Tay Y, Zhang J, Thomson AM, Lim B, Rigoutsos I (2008) MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 455(7216):1124–1128. doi:10.1038/nature07299

    Article  CAS  Google Scholar 

  3. Bartels CL, Tsongalis GJ (2009) MicroRNAs: novel biomarkers for human cancer. Clin Chem 55(4):623–631. doi:10.1373/clinchem.2008.112805

    Article  CAS  Google Scholar 

  4. Kato M, Castro NE, Natarajan R (2013) MicroRNAs: potential mediators and biomarkers of diabetic complications. Free Radical Bio Med 64:85–94. doi:10.1016/j.freeradbiomed.2013.06.009

    Article  CAS  Google Scholar 

  5. Cortez MA, Bueso-Ramos C, Ferdin J, Lopez-Berestein G, Sood AK, Calin GA (2011) MicroRNAs in body fluids-the mix of hormones and biomarkers. Nat Rev Clin Oncol 8(8):467–477. doi:10.1038/nrclinonc.2011.76

    Article  CAS  Google Scholar 

  6. Dong H, Lei J, Ding L, Wen Y, Ju H, Zhang X (2013) MicroRNA: function, detection, and bioanalysis. Chem Rev 113(8):6207–6233. doi:10.1021/cr300362f

    Article  CAS  Google Scholar 

  7. Li W, Hou T, Wu M, Li F (2016) Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide. Talanta 148:116–121. doi:10.1016/j.talanta.2015.10.078

    Article  CAS  Google Scholar 

  8. Sang Y, Xu Y, Xu L, Cheng W, Li X, Wu J, Ding S (2017) Colorimetric and visual determination of microRNA via cycling signal amplification using T7 exonuclease. Microchim Acta 184(7):2465–2471. doi:10.1007/s00604-017-2238-8

    Article  CAS  Google Scholar 

  9. Xu Y, Li D, Cheng W, Hu R, Sang Y, Yin Y, Ding S, Ju H (2016) Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machinery. Anal Chim Acta 936:229–235. doi:10.1016/j.aca.2016.07.007

    Article  CAS  Google Scholar 

  10. Zeng K, Li H, Peng Y (2017) Gold nanoparticle enhanced surface plasmon resonance imaging of microRNA-155 using a functional nucleic acid-based amplification machine. Microchim Acta. doi:10.1007/s00604-017-2276-2

  11. Zhang H, Liu Y, Fu X, Yuan L, Zhu Z (2015) Microfluidic bead-based assay for microRNAs using quantum dots as labels and enzymatic amplification. Microchim Acta 182(3):661–669. doi:10.1007/s00604-014-1372-9

    Article  CAS  Google Scholar 

  12. Zhou L, Wang J, Chen Z, Li J, Wang T, Zhang Z, Xie G (2017) A universal electrochemical biosensor for the highly sensitive determination of microRNAs based on isothermal target recycling amplification and a DNA signal transducer triggered reaction. Microchim Acta 184(5):1305–1313. doi:10.1007/s00604-017-2129-z

    Article  CAS  Google Scholar 

  13. Liu H, Bei X, Xia Q, Fu Y, Zhang S, Liu M, Fan K, Zhang M, Yang Y (2016) Enzyme-free electrochemical detection of microRNA-21 using immobilized hairpin probes and a target-triggered hybridization chain reaction amplification strategy. Microchim Acta 183(1):297–304. doi:10.1007/s00604-015-1636-z

    Article  CAS  Google Scholar 

  14. Yang J, Tang M, Diao W, Cheng W, Zhang Y, Yan Y (2016) Electrochemical strategy for ultrasensitive detection of microRNA based on MNAzyme-mediated rolling circle amplification on a gold electrode. Microchim Acta 183(11):3061–3067. doi:10.1007/s00604-016-1958-5

    Article  CAS  Google Scholar 

  15. Hou T, Li W, Liu X, Li F (2015) Label-free and enzyme-free homogeneous electrochemical biosensing strategy based on hybridization chain reaction: a facile, sensitive, and highly specific microRNA assay. Anal Chem 87(22):11368–11374. doi:10.1021/acs.analchem.5b02790

    Article  CAS  Google Scholar 

  16. Wang M, Yang Z, Guo Y, Wang X, Yin H, Ai S (2015) Visible-light induced photoelectrochemical biosensor for the detection of microRNA based on Bi2S3 nanorods and streptavidin on an ITO electrode. Microchim Acta 182(1):241–248. doi:10.1007/s00604-014-1324-4

    Article  CAS  Google Scholar 

  17. Yu Y-Q, Wang J-P, Zhao M, Hong L-R, Chai Y-Q, Yuan R, Zhuo Y (2016) Target-catalyzed hairpin assembly and intramolecular/intermolecular co-reaction for signal amplified electrochemiluminescent detection of microRNA. Biosens Bioelectron 77:442–450. doi:10.1016/j.bios.2015.09.056

    Article  CAS  Google Scholar 

  18. Liu T, Chen X, Hong C-Y, Xu X-P, Yang H-H (2014) Label-free and ultrasensitive electrochemiluminescence detection of microRNA based on long-range self-assembled DNA nanostructures. Microchim Acta 181(7):731–736. doi:10.1007/s00604-013-1113-5

    Article  CAS  Google Scholar 

  19. Liu H, Tian T, Zhang Y, Ding L, Yu J, Yan M (2017) Sensitive and rapid detection of microRNAs using hairpin probes-mediated exponential isothermal amplification. Biosens Bioelectron 89:710–714. doi:10.1016/j.bios.2016.10.099

    Article  Google Scholar 

  20. Nie J, Zhang D-W, Tie C, Zhou Y-L, Zhang X-X (2014) G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection. Biosens Bioelectron 56:237–242. doi:10.1016/j.bios.2014.01.032

    Article  CAS  Google Scholar 

  21. Tan E, Wong J, Nguyen D, Zhang Y, Erwin B, Van Ness LK, Baker SM, Galas DJ, Niemz A (2005) Isothermal DNA amplification coupled with DNA nanosphere-based colorimetric detection. Anal Chem 77(24):7984–7992. doi:10.1021/ac051364i

    Article  CAS  Google Scholar 

  22. Dong H, Zhang J, Ju H, Lu H, Wang S, Jin S, Hao K, Du H, Zhang X (2012) Highly sensitive multiple microRNA detection based on fluorescence quenching of graphene oxide and isothermal strand-displacement polymerase reaction. Anal Chem 84(10):4587–4593. doi:10.1021/ac300721u

    Article  CAS  Google Scholar 

  23. Liu H, Li L, Duan L, Wang X, Xie Y, Tong L, Wang Q, Tang B (2013) High specific and ultrasensitive isothermal detection of microRNA by padlock probe-based exponential rolling circle amplification. Anal Chem 85(16):7941–7947. doi:10.1021/ac401715k

    Article  CAS  Google Scholar 

  24. Xu Z, Yin H, Tian Z, Zhou Y, Ai S (2014) Electrochemical immunoassays for the detection the activity of DNA methyltransferase by using the rolling circle amplification technique. Microchim Acta 181(3):471–477. doi:10.1007/s00604-013-1141-1

    Article  CAS  Google Scholar 

  25. Shen B, Li J, Cheng W, Yan Y, Tang R, Li Y, Ju H, Ding S (2015) Electrochemical aptasensor for highly sensitive determination of cocaine using a supramolecular aptamer and rolling circle amplification. Microchim Acta 182(1):361–367. doi:10.1007/s00604-014-1333-3

    Article  CAS  Google Scholar 

  26. Gu H, Hao L, Duan N, Wu S, Xia Y, Ma X, Wang Z (2017) A competitive fluorescent aptasensor for okadaic acid detection assisted by rolling circle amplification. Microchim Acta. doi:10.1007/s00604-017-2293-1

  27. Rafiee-Pour H-A, Behpour M, Keshavarz M (2016) A novel label-free electrochemical miRNA biosensor using methylene blue as redox indicator: application to breast cancer biomarker miRNA-21. Biosens Bioelectron 77:202–207. doi:10.1016/j.bios.2015.09.025

    Article  CAS  Google Scholar 

  28. Huang YL, Mo S, Gao ZF, Chen JR, Lei JL, Luo HQ, Li NB (2017) Amperometric biosensor for microRNA based on the use of tetrahedral DNA nanostructure probes and guanine nanowire amplification. Microchim Acta. doi:10.1007/s00604-017-2246-8

  29. Chi B-Z, Liang R-P, Qiu W-B, Yuan Y-H, Qiu J-D (2017) Direct fluorescence detection of microRNA based on enzymatically engineered primer extension poly-thymine (EPEPT) reaction using copper nanoparticles as nano-dye. Biosens Bioelectron 87:216–221. doi:10.1016/j.bios.2016.08.042

    Article  CAS  Google Scholar 

  30. Lv W, Zhao J, Situ B, Li B, Ma W, Liu J, Wu Z, Wang W, Yan X, Zheng L (2016) A target-triggered dual amplification strategy for sensitive detection of microRNA. Biosens Bioelectron 83:250–255. doi:10.1016/j.bios.2016.04.053

    Article  CAS  Google Scholar 

  31. Niu C, Song Q, He G, Na N, Ouyang J (2016) Near-infrared-fluorescent probes for bioapplications based on silica-coated gold nanobipyramids with distance-dependent plasmon-enhanced fluorescence. Anal Chem 88(22):11062–11069. doi:10.1021/acs.analchem.6b03034

    Article  CAS  Google Scholar 

  32. Yin H, Zhou Y, Li B, Li X, Yang Z, Ai S, Zhang X (2016) Photoelectrochemical immunosensor for microRNA detection based on gold nanoparticles-functionalized g-C3N4 and anti-DNA:RNA antibody. Sens Actuators, B-Chem 222:1119–1126. doi:10.1016/j.snb.2015.08.019

    Article  CAS  Google Scholar 

  33. Li B, Li X, Wang M, Yang Z, Yin H, Ai S (2015) Photoelectrochemical biosensor for highly sensitive detection of microRNA based on duplex-specific nuclease-triggered signal amplification. J Solid State Electrochem 19(5):1301–1309. doi:10.1007/s10008-015-2747-5

    Article  CAS  Google Scholar 

  34. Chen Y, Xiang Y, Yuan R, Chai Y (2015) Intercalation of quantum dots as the new signal acquisition and amplification platform for sensitive electrochemiluminescent detection of microRNA. Anal Chim Acta 891:130–135. doi:10.1016/j.aca.2015.07.059

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Natural Science Foundation of Shandong province, China (No. ZR2014BQ029, ZR2017MD023), the National Natural Science Foundation of China (No. 21375079), and the National Key Research and Development Program of China (2016YFD0800304).

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Authors and Affiliations

  1. College of Chemistry and Material Science, Shandong Agricultural University, Taian, 271018, People’s Republic of China

    Yunlei Zhou, Bingchen Li, Minghui Wang, Huanshun Yin & Shiyun Ai

  2. College of Resources and Environment, Key Laboratory of Agricultural Environment in Universities of Shandong, Shandong Agricultural University, Taian, 271018, People’s Republic of China

    Jun Wang

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  1. Yunlei Zhou
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  2. Bingchen Li
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  3. Minghui Wang
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  4. Jun Wang
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Correspondence to Jun Wang or Huanshun Yin.

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Zhou, Y., Li, B., Wang, M. et al. Fluorometric determination of microRNA based on strand displacement amplification and rolling circle amplification. Microchim Acta 184, 4359–4365 (2017). https://doi.org/10.1007/s00604-017-2450-6

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