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.