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Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis

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Abstract

Development of a rapid and accurate quantification method for the detection of microRNAs (miRNAs) has been desired, in particular, when they are differently expressed in normal and pathological conditions. However, various methods for the quantification of small non-coding RNAs as well as miRNAs have been described. These methods mainly include hybridization-based approaches such as primer extension, northern blotting, microarray profiling, and reverse transcription (RT) PCR. Here, we developed a simple and rapid method based on stem-loop primer-based real-time PCR assay for sensitive and accurate detection of mature miRNAs. Initially, a miRNA-specific stem-loop RT primer is used for RT, which is followed by TaqMan real-time PCR assay using specific forward primer in combination with universal reverse primer and TaqMan probe. The assay has shown high sensitivity (≤50 copies/reaction) for miRNA detection in two breast cancer cell lines, MCF-7 and MDA-MB-231. This assay might be implicated as a rapid and cost effective method for the detection of small non-coding RNAs.

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References

  1. Bartels CL, Tsongalis GJ (2009) MicroRNAs: novel biomarkers for human cancer. Clin Chem 55(4):623–631

    Article  PubMed  CAS  Google Scholar 

  2. Benes V, Castoldi M (2010) Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50(4):244–249

    Article  PubMed  CAS  Google Scholar 

  3. Chen Y, Gelfond JA, McManus LM, Shireman PK (2009) Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis. BMC Genomics 10:407

    Article  PubMed  Google Scholar 

  4. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179

    Article  PubMed  Google Scholar 

  5. Barad O, Meiri E, Avniel A, Aharonov R, Barzilai A, Bentwich I, Einav U, Gilad S, Hurban P, Karov Y, Lobenhofer EK, Sharon E, Shiboleth YM, Shtutman M, Bentwich Z, Einat P (2004) MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues. Genome Res 14(12):2486–2494

    Article  PubMed  CAS  Google Scholar 

  6. Watanabe Y, Tomita M, Kanai A (2007) Computational methods for microRNA target prediction. Methods Enzymol 427:65–86

    Article  PubMed  CAS  Google Scholar 

  7. Gusev Y (2008) Computational methods for analysis of cellular functions and pathways collectively targeted by differentially expressed microRNA. Methods 44(1):61–72

    Article  PubMed  CAS  Google Scholar 

  8. Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15–20

    Article  PubMed  CAS  Google Scholar 

  9. Grun D, Wang YL, Langenberger D, Gunsalus KC, Rajewsky N (2005) microRNA target predictions across seven Drosophila species and comparison to mammalian targets. PLoS Comput Biol 1(1):e13

    Article  PubMed  Google Scholar 

  10. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS (2003) MicroRNA targets in Drosophila. Genome Biol 5(1):R1

    Article  PubMed  Google Scholar 

  11. Maragkakis M, Alexiou P, Papadopoulos GL, Reczko M, Dalamagas T, Giannopoulos G, Goumas G, Koukis E, Kourtis K, Simossis VA, Sethupathy P, Vergoulis T, Koziris N, Sellis T, Tsanakas P, Hatzigeorgiou AG (2009) Accurate microRNA target prediction correlates with protein repression levels. BMC Bioinformatics 10:295

    Article  PubMed  Google Scholar 

  12. Han J, Hendzel MJ, Allalunis-Turner J (2011) Notch signaling as a therapeutic target for breast cancer treatment? Breast Cancer Res 13(3):210

    Article  PubMed  Google Scholar 

  13. Liu S (2011) The ROCK signaling and breast cancer metastasis. Mol Biol Rep 38(2):1363–1366

    Article  PubMed  CAS  Google Scholar 

  14. Yamazaki D, Kurisu S, Takenawa T (2009) Involvement of Rac and Rho signaling in cancer cell motility in 3D substrates. Oncogene 28(13):1570–1583

    Article  PubMed  CAS  Google Scholar 

  15. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36

    Article  PubMed  Google Scholar 

  16. Castoldi M, Schmidt S, Benes V, Hentze MW, Muckenthaler MU (2008) miChip: an array-based method for microRNA expression profiling using locked nucleic acid capture probes. Nat Protoc 3(2):321–329

    Article  PubMed  CAS  Google Scholar 

  17. Huang Y, Zou Q, Wang SP, Tang SM, Zhang GZ, Shen XJ (2011) The discovery approaches and detection methods of microRNAs. Mol Biol Rep 38(6):4125–4135

    Article  PubMed  CAS  Google Scholar 

  18. Chen C, Tan R, Wong L, Fekete R, Halsey J (2011) Quantitation of microRNAs by real-time RT-qPCR. Methods Mol Biol 687:113–134

    Article  PubMed  CAS  Google Scholar 

  19. Hurley J, Roberts D, Bond A, Keys D, Chen C (2012) Stem-loop RT-qPCR for microRNA expression profiling. Methods Mol Biol 822:33–52

    Article  PubMed  CAS  Google Scholar 

  20. Liu CG, Calin GA, Meloon B, Gamliel N, Sevignani C, Ferracin M, Dumitru CD, Shimizu M, Zupo S, Dono M, Alder H, Bullrich F, Negrini M, Croce CM (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci USA 101(26):9740–9744

    Article  PubMed  CAS  Google Scholar 

  21. Mestdagh P, Feys T, Bernard N, Guenther S, Chen C, Speleman F, Vandesompele J (2008) High throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. Nucleic Acids Res 36(21):e143

    Article  PubMed  Google Scholar 

  22. Doran J, Strauss WM (2007) Bio-informatic trends for the determination of miRNA-target interactions in mammals. DNA Cell Biol 26(5):353–360

    Article  PubMed  CAS  Google Scholar 

  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods 25(4):402–408

    Article  PubMed  CAS  Google Scholar 

  24. Li L, Xie X, Luo J, Liu M, Xi S, Guo J, Kong Y, Wu M, Gao J, Xie Z, Tang J, Wang X, Wei W, Yang M, Hung MC (2012) Targeted expression of miR-34a using the T-VISA system suppresses breast cancer cell growth and invasion. Mol Ther 20(12):2326–2334

    Article  PubMed  CAS  Google Scholar 

  25. Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H, Taylor M, Harris AL, Ragoussis J (2011) MicroRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Cancer Res 71(17):5635–5645

    Article  PubMed  CAS  Google Scholar 

  26. Valastyan S, Reinhardt F, Benaich N, Calogrias D, Szasz AM, Wang ZC, Brock JE, Richardson AL, Weinberg RA (2009) A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis. Cell 137(6):1032–1046

    Article  PubMed  CAS  Google Scholar 

  27. Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini M, Croce CM (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 101(9):2999–3004

    Article  PubMed  CAS  Google Scholar 

  28. Li W, Ruan K (2009) MicroRNA detection by microarray. Anal Bioanal Chem 394(4):1117–1124

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was funded by Pasteur Institute of Iran, Tehran. The authors appreciate Stem Cell Technology Research Center, Tehran, Iran, for providing technical support. The authors would like to thank Dr. Houri Rezvan, Dr. Hossein Ghanbarian and Zahra Masoumi for final edition on the manuscript.

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Authors and Affiliations

  1. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, P.O. Box 1316943551, Tehran, Iran

    Samira Mohammadi-Yeganeh, Mahdi Paryan, Reza Mahdian & Morteza Karimipoor

  2. Food and Drug Laboratory Research Center, Ministry of Health and Medical Education, Tehran, Iran

    Siamak Mirab Samiee

  3. Department of Hematology, Tarbiat Modares University, Tehran, Iran

    Masoud Soleimani

  4. Department of Molecular Biology and Genetic Engineering, Stem Cell Technology Research Center, Tehran, Iran

    Masoud Soleimani & Ehsan Arefian

  5. Department of Virology, Pasteur Institute of Iran, Tehran, Iran

    Keyhan Azadmanesh

  6. Department of Epidemiology, Pasteur Institute of Iran, Tehran, Iran

    Ehsan Mostafavi

Authors
  1. Samira Mohammadi-Yeganeh
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  2. Mahdi Paryan
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  3. Siamak Mirab Samiee
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  4. Masoud Soleimani
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  7. Ehsan Mostafavi
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  8. Reza Mahdian
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  9. Morteza Karimipoor
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Corresponding authors

Correspondence to Reza Mahdian or Morteza Karimipoor.

Electronic supplementary material

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11033_2012_2442_MOESM1_ESM.doc

Supplementary Table 1: Comparison between some commercial kits and our developed technique in format and price (DOC 33 kb)

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Mohammadi-Yeganeh, S., Paryan, M., Mirab Samiee, S. et al. Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis. Mol Biol Rep 40, 3665–3674 (2013). https://doi.org/10.1007/s11033-012-2442-x

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  • DOI: https://doi.org/10.1007/s11033-012-2442-x

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