Abstract
Small non-coding RNAs are considered be involved in the regulation of multiple cellular processes. Quantitative reverse transcription PCR (RT-qPCR) is widely used in the detection of eukaryotic microRNA, and the stem-loop primers can improve the specificity and efficiency of reverse transcription. However, the loop structure of primers probably influence the next quantitative amplification due to the base stacking and steric hindrance. Here, we designed a chimeric stem-loop primer with a deoxyuracil (dU) base located near the RNA matching part. After the reverse transcription, uracil-DNA glycosylase (UDG) treatment was used to remove the dU base and destroy the stem-loop structure of RT product. Enzymatic assay confirmed that the recombinant UDG could efficiently eliminate the dU base in the oligonucleotide. Transcriptions of two small RNAs (TFF and ryeA) in Escherichia coli were detected by RT-qPCR with different primers. Results showed that the use of the chimeric dU stem-loop primer and UDG treatment could enhance the detection specificity and sensitivity about 1.1- to 3.4-fold, compared to those with traditional stem-loop primer and linear primer. Total RNA of 1–10 pg was enough for efficient detection with the chimeric stem-loop primers. In a word, this strategy could promote the RT-qPCR detection efficiency on the transcription of bacterial small RNAs even in trace samples and can facilitate the detection of exiguous change in cellular metabolism.
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References
Altuvia S (2007) Identification of bacterial small non-coding RNAs: experimental approaches. Curr Opin in Microbiol 10:257–261
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:e179
Chen J, Lozach J, Garcia EW, Barnes B, Luo S, Mikoulitch I, Zhou L, Schroth G, Fan JB (2008) Highly sensitive and specific microRNA expression profiling using BeadArray technology. Nucleic Acids Res 36:e87
Cheng A, Li M, Liang Y, Wang Y, Wong L, Chen C, Vlassov AV, Magdaleno S (2009) Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo. Oligonucleotides 19:203–208
Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, Green R, Meltzer PS, Wolfsberg TG, Collins FS (2006) DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 3:503–509
Czimmerer Z, Hulvely J, Simandi Z, Varallyay E, Havelda Z, Szabo E, Varga A, Dezso B, Balogh M, Horvath A, Domokos B, Torok Z, Nagy L, Balint BL (2013) A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules. PLoS One 8:e55168
Dellett M, Simpson DA (2016) Considerations for optimization of microRNA PCR assays for molecular diagnosis. Expert Rev Mol Diagn 16(4):407–414
Hershberg R, Altuvia S, Margalit H (2003) A survey of small RNA-encoding genes in Escherichia coli. Nucleic Acids Res 31:1813–1820
Huang HY, Chang HY, Chou CH, Tseng CP, Ho SY, Yang CD, Ju YW, Huang HD (2009) sRNAMap: genomic maps for small non-coding RNAs, their regulators and their targets in microbial genomes. Nucleic Acids Res 37(Database issue):D150–D154
Krichevsky AM, King KS, Donahue CP, Khrapko K, Kosik KS (2003) A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9:1274–1281
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858
Lim LP, Lau NC, Weinstein EG, Abdelhakim A, Yekta S, Rhoades MW, Burge CB, Bartel DP (2003) The microRNAs of Caenorhabditis elegans. Genes Dev 17:991–1008
Liu X, Liu J (2004) Cloning, expression, and characterization of uracil-DNA glycosylase of Chlamydia pneumoniae in Escherichia coli. Protein Expr Purif 35:46–53
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 U S A 101:9740–9704
Liu XP, Li CP, Hou JL, Liu YF, Liang RB, Liu JH (2010) Expression and characterization of thymine-DNA glycosylase from Aeropyrum pernix. Protein Expr Purif 70:1–6
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 25:402–408
Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ (2005) Elucidation of the small RNA component of the transcriptome. Science 309:1567–1569
Marcial-Quino J, Gómez-Manzo S, Fierro F, Vanoye-Carlo A, Rufino-González Y, Sierra-Palacios E, Castillo-Villanueva A, Castillo-Rodríguez RA, Rodríguez-Bustamante E, Arreguin-Espinosa R, Reyes-Vivas H. (2016) Stem-loop RT-qPCR as an efficient tool for the detection and quantification of small RNAs in Giardia lamblia. Genes (Basel).7(12)
Pandey SP, Winkler JA, Li H, Camacho DM, Collins JJ, Walker GC (2014) Central role for RNase YbeY in Hfq-dependent and Hfq-independent small-RNA regulation in bacteria. BMC Genomics 15:121
Pena JT, Sohn-Lee C, Rouhanifard SH, Ludwig J, Hafner M, Mihailovic A, Lim C, Holoch D, Berninger P, Zavolan M, Tuschl T (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141
Shen WH, Hohn B (1992) DMSO improves PCR amplification of DNA with complex secondary structure. Trends Genet 8(7):228
Smith C, Day PJ, Walker MR (1993) Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. PCR Methods Appl 2:328–332
Varkonyi-Gasic E (2017) Stem-loop qRT-PCR for the detection of plant microRNAs. Methods Mol Biol 1456:163–175
Vogel J, Sharma CM (2005) How to find small non-coding RNAs in bacteria. Biol Chem 386(12):1219–1238
Vogel J, Wagner EG (2007) Target identification of small noncoding RNAs in bacteria. Curr Opin Microbiol 10(3):262–270
Wagner M, van Wolferen M, Wagner A, Lassak K, Meyer BH, Reimann J, Albers SV (2012) Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius. Front Microbiol 3:214
Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136:615–628
Yang LH, Wang SL, Tang LL, Liu B, Ye WL, Wang LL, Wang ZY, Zhou MT, Chen BC (2014) Universal stem-loop primer method for screening and quantification of microRNA. PLoS One 9:e115293
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97:5978–5983
Zeng Y, Cullen BR (2003) Sequence requirements for micro RNA processing and function in human cells. RNA 9:112–123
Authors’ contributions
YW performed the experiments and RL and JL designed the experiments. YW and RL analyzed the data and wrote the manuscript. XX and TY assisted with the experiments.
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This study was funded by the National Science Foundation of China (Grant No. 31370152, 31570099), the Shanghai Pujiang Program (14PJD020) and the Chen Xing Grant of Shanghai Jiaotong University.
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The authors declare that they have no conflict of interest.
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This article does not contain any studies with human participants or animals performed by any of the authors.
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Wu, Y., Xing, X., You, T. et al. RT-qPCR with chimeric dU stem-loop primer is efficient for the detection of bacterial small RNAs. Appl Microbiol Biotechnol 101, 4561–4568 (2017). https://doi.org/10.1007/s00253-017-8181-0
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DOI: https://doi.org/10.1007/s00253-017-8181-0