Published online 27 November 2005
Methods Online |
Real-time quantification of microRNAs by stem–loop RT–PCR
Applied Biosystems 850 Lincoln Centre Drive, Foster City, CA 94404, USA
*To whom correspondence should be addressed. Tel: +1 650 638 5245; Fax: +1 650 638 6343; Email: chencx{at}appliedbiosystems.com
Received May 24, 2005. Revised July 8, 2005. Accepted October 25, 2005.
A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
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R. Garzon, S. Volinia, C.-G. Liu, C. Fernandez-Cymering, T. Palumbo, F. Pichiorri, M. Fabbri, K. Coombes, H. Alder, T. Nakamura, et al. MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia Blood, March 15, 2008; 111(6): 3183 - 3189. [Abstract] [Full Text] [PDF]
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R. Garzon, M. Garofalo, M. P. Martelli, R. Briesewitz, L. Wang, C. Fernandez-Cymering, S. Volinia, C.-G. Liu, S. Schnittger, T. Haferlach, et al. Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin PNAS, March 11, 2008; 105(10): 3945 - 3950. [Abstract] [Full Text] [PDF]
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K. Hosoda, T. Matsuura, H. Kita, N. Ichihashi, K. Tsukada, I. Urabe, and T. Yomo A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA RNA, March 1, 2008; 14(3): 584 - 592. [Abstract] [Full Text] [PDF]
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M. Bak, A. Silahtaroglu, M. Moller, M. Christensen, M. F. Rath, B. Skryabin, N. Tommerup, and S. Kauppinen MicroRNA expression in the adult mouse central nervous system RNA, March 1, 2008; 14(3): 432 - 444. [Abstract] [Full Text] [PDF]
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S. S.C. Chim, T. K.F. Shing, E. C.W. Hung, T.-y. Leung, T.-k. Lau, R. W.K. Chiu, and Y.M. Dennis Lo Detection and Characterization of Placental MicroRNAs in Maternal Plasma Clin. Chem., March 1, 2008; 54(3): 482 - 490. [Abstract] [Full Text] [PDF]
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J. Jiang, Y. Gusev, I. Aderca, T. A. Mettler, D. M. Nagorney, D. J. Brackett, L. R. Roberts, and T. D. Schmittgen Association of MicroRNA Expression in Hepatocellular Carcinomas with Hepatitis Infection, Cirrhosis, and Patient Survival Clin. Cancer Res., January 15, 2008; 14(2): 419 - 427. [Abstract] [Full Text] [PDF]
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Q. Wang, Z. Huang, H. Xue, C. Jin, X.-L. Ju, J.-D. J. Han, and Y.-G. Chen MicroRNA miR-24 inhibits erythropoiesis by targeting activin type I receptor ALK4 Blood, January 15, 2008; 111(2): 588 - 595. [Abstract] [Full Text] [PDF]
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Y. Guo, Z. Chen, L. Zhang, F. Zhou, S. Shi, X. Feng, B. Li, X. Meng, X. Ma, M. Luo, et al. Distinctive MicroRNA Profiles Relating to Patient Survival in Esophageal Squamous Cell Carcinoma Cancer Res., January 1, 2008; 68(1): 26 - 33. [Abstract] [Full Text] [PDF]
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E. J. Lee, M. Baek, Y. Gusev, D. J. Brackett, G. J. Nuovo, and T. D. Schmittgen Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors RNA, January 1, 2008; 14(1): 35 - 42. [Abstract] [Full Text] [PDF]
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I. Ibarra, Y. Erlich, S. K. Muthuswamy, R. Sachidanandam, and G. J. Hannon A role for microRNAs in maintenance of mouse mammary epithelial progenitor cells Genes & Dev., December 15, 2007; 21(24): 3238 - 3243. [Abstract] [Full Text] [PDF]
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B. D. Brown, A. Cantore, A. Annoni, L. S. Sergi, A. Lombardo, P. Della Valle, A. D'Angelo, and L. Naldini A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice Blood, December 15, 2007; 110(13): 4144 - 4152. [Abstract] [Full Text] [PDF]
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S. Mi, J. Lu, M. Sun, Z. Li, H. Zhang, M. B. Neilly, Y. Wang, Z. Qian, J. Jin, Y. Zhang, et al. MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia PNAS, December 11, 2007; 104(50): 19971 - 19976. [Abstract] [Full Text] [PDF]
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J. Huang, Z. Liang, B. Yang, H. Tian, J. Ma, and H. Zhang Derepression of MicroRNA-mediated Protein Translation Inhibition by Apolipoprotein B mRNA-editing Enzyme Catalytic Polypeptide-like 3G (APOBEC3G) and Its Family Members J. Biol. Chem., November 16, 2007; 282(46): 33632 - 33640. [Abstract] [Full Text] [PDF]
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Y. K. Tong, R. W.K. Chiu, T. Y. Leung, C. Ding, T. K. Lau, T. N. Leung, and Y.M. D. Lo Detection of Restriction Enzyme Digested Target DNA by PCR Amplification Using a Stem-Loop Primer: Application to the Detection of Hypomethylated Fetal DNA in Maternal Plasma Clin. Chem., November 1, 2007; 53(11): 1906 - 1914. [Abstract] [Full Text] [PDF]
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M. Fabbri, R. Garzon, A. Cimmino, Z. Liu, N. Zanesi, E. Callegari, S. Liu, H. Alder, S. Costinean, C. Fernandez-Cymering, et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B PNAS, October 2, 2007; 104(40): 15805 - 15810. [Abstract] [Full Text] [PDF]
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Y. Xi, G. Nakajima, E. Gavin, C. G. Morris, K. Kudo, K. Hayashi, and J. Ju Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples RNA, October 1, 2007; 13(10): 1668 - 1674. [Abstract] [Full Text] [PDF]
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D. C. Corney, A. Flesken-Nikitin, A. K. Godwin, W. Wang, and A. Yu. Nikitin MicroRNA-34b and MicroRNA-34c Are Targets of p53 and Cooperate in Control of Cell Proliferation and Adhesion-Independent Growth Cancer Res., September 15, 2007; 67(18): 8433 - 8438. [Abstract] [Full Text] [PDF]
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