title: Simple and sensitive microRNA labeling by terminal deoxynucleotidyl transferase.
abstract: MicroRNAs (miRNAs) constitute a critically important class of non-translated, small RNAs, which post-transcriptionally regulate gene expression via one of the multiple mechanisms. To profile miRNA expression, microarrays have been extensively applied to the high-throughput detection of miRNAs. Here, we described a novel 3'-end miRNA-labeling method for microarray detection by terminal-deoxynucleotidyl transferase (TdT). TdT can catalyze the formation of polynucleotides at RNA receptor molecule with deoxycytidine triphosphate (dCTP). Using this activity, miRNA was successfully labeled by adding fluorescence dCTPs to its 3'-end. This labeling method was very simple and sensitive. The TdT-labeling method can detect as little as 0.04 fmol of synthetic small RNA, and produce precise and accurate measurements that span a linear dynamic range from 0.04 to 5 fmol of synthetic small RNA. The high consistency of miRNA expression data between our TdT method and real-time polymerase chain reaction analysis indicated the reliability and accuracy of the TdT method. Taken together, these results emphasize the immense potential application of the TdT-labeling method for sensitive and high-throughput microarray analysis of miRNA expression.
authors.affiliation: School of Life Sciences, Shanghai University, China. botaozhao@gmail.com
authors.forename: Botao
authors.initials: B
authors.lastname: Zhao
authors.forename: Zizhen
authors.initials: Z
authors.lastname: Gong
authors.forename: Zhizheng
authors.initials: Z
authors.lastname: Gong
authors.forename: Zhongliang
authors.initials: Z
authors.lastname: Ma
authors.forename: Detao
authors.initials: D
authors.lastname: Wang
authors.forename: Youxin
authors.initials: Y
authors.lastname: Jin
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004253:DNA Nucleotidylexotransferase; D015342:DNA Probes / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; D004253:DNA Nucleotidylexotransferase