Abstract
Cellular RNAs are often expressed as multiple isoforms of complex heterogeneity in both length and terminal sequences. IsomiRs, the isoforms of microRNAs, are such an example. Distinct quantification of each RNA variant is necessary to unravel the biogenesis mechanism and biological significance of heterogenetic RNA expression. Here we describe Dumbbell-PCR (Db-PCR), a TaqMan RT-PCR-based method that distinctively quantifies a specific small RNA variant with single-nucleotide resolution at terminal sequences. Db-PCR enables the quantitative analysis of RNA terminal heterogeneity without performing Next-Generation Sequencing.
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References
Neilsen CT, Goodall GJ, Bracken CP (2012) IsomiRs–the overlooked repertoire in the dynamic microRNAome. Trends Genet 28(11):544–549. doi:10.1016/j.tig.2012.07.005. S0168-9525(12)00112-6 [pii]
Somel M, Guo S, Fu N, Yan Z, Hu HY, Xu Y, Yuan Y, Ning Z, Hu Y, Menzel C, Hu H, Lachmann M, Zeng R, Chen W, Khaitovich P (2010) MicroRNA, mRNA, and protein expression link development and aging in human and macaque brain. Genome Res 20(9):1207–1218. doi:10.1101/gr.106849.110. gr.106849.110 [pii]
Fernandez-Valverde SL, Taft RJ, Mattick JS (2010) Dynamic isomiR regulation in drosophila development. RNA 16(10):1881–1888. doi:10.1261/rna.2379610. rna.2379610 [pii]
Bizuayehu TT, Lanes CF, Furmanek T, Karlsen BO, Fernandes JM, Johansen SD, Babiak I (2012) Differential expression patterns of conserved miRNAs and isomiRs during Atlantic halibut development. BMC Genomics 13:11. doi:10.1186/1471-2164-13-11. 1471-2164-13-11 [pii]
Tan GC, Chan E, Molnar A, Sarkar R, Alexieva D, Isa IM, Robinson S, Zhang S, Ellis P, Langford CF, Guillot PV, Chandrashekran A, Fisk NM, Castellano L, Meister G, Winston RM, Cui W, Baulcombe D, Dibb NJ (2014) 5′ isomiR variation is of functional and evolutionary importance. Nucleic Acids Res 42(14):9424–9435. doi:10.1093/nar/gku656. gku656 [pii]
Loher P, Londin ER, Rigoutsos I (2014) IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies. Oncotarget 5(18):8790–8802. doi:10.18632/oncotarget.2405. 2405 [pii]
Telonis AG, Loher P, Jing Y, Londin E, Rigoutsos I (2015) Beyond the one-locus-one-miRNA paradigm: microRNA isoforms enable deeper insights into breast cancer heterogeneity. Nucleic Acids Res 43(19):9158–9175. doi:10.1093/nar/gkv922. gkv922 [pii]
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. doi:10.1093/nar/gni178. 33/20/e179 [pii]
Schamberger A, Orban TI (2014) 3′ IsomiR species and DNA contamination influence reliable quantification of microRNAs by stem-loop quantitative PCR. PLoS One 9(8):e106315. doi:10.1371/journal.pone.0106315. PONE-D-13-49830 [pii]
Honda S, Kirino Y (2015) Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences. Nucleic Acids Res 43(12):e77. doi:10.1093/nar/gkv218. gkv218 [pii]
Bullard DR, Bowater RP (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem J 398(1):135–144. doi:10.1042/BJ20060313. BJ20060313 [pii]
Nandakumar J, Ho CK, Lima CD, Shuman S (2004) RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2. J Biol Chem 279(30):31337–31347. doi:10.1074/jbc.M402394200. M402394200 [pii]
Nandakumar J, Shuman S (2005) Dual mechanisms whereby a broken RNA end assists the catalysis of its repair by T4 RNA ligase 2. J Biol Chem 280(25):23484–23489. doi:10.1074/jbc.M500831200. M500831200 [pii]
Ranade K, Chang MS, Ting CT, Pei D, Hsiao CF, Olivier M, Pesich R, Hebert J, Chen YD, Dzau VJ, Curb D, Olshen R, Risch N, Cox DR, Botstein D (2001) High-throughput genotyping with single nucleotide polymorphisms. Genome Res 11(7):1262–1268. doi:10.1101/gr.157801
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31(13):3406–3415
Acknowledgments
This study was supported by NIH grant (GM106047 to YK).
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Shigematsu, M., Honda, S., Kirino, Y. (2018). Dumbbell-PCR for Discriminative Quantification of a Small RNA Variant. In: Okamura, K., Nakanishi, K. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 1680. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7339-2_4
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DOI: https://doi.org/10.1007/978-1-4939-7339-2_4
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