Abstract: The indigenous small non-coding RNAs, known as microRNAs (miRNAs), are important regulators of gene expression and many of them are evolutionarily conserved. Whether stem-loop RT-PCR, as a sensitive method, could be utilized to clone conserved miRNAs from non-model insects lacks information. Here, three miRNAs, sli-miR-14, sli-miR-2a and sli-bantam, were cloned from Spodoptera litura by stem-loop RT-PCR. Two groups of primers were designed, and one of them performed especially well and proved stable. The sequences of two highly conserved miRNAs, sli-miR-14 and sli-miR-2a were identical to those in Drosophila melanogaster. To validate the reliability of this strategy, pre-miR-14 and pre-miR-2a in S. litura as representatives were given as well; this shared high homology with those in D. melanogaster and Bombyx mori, and both mature sequences of sli-miR-14 and sli-miR-2a in their precursors shared 100% identity to the results shown by stem-loop RT-PCR. Moreover, expression patterns of these miRNAs were investigated by real-time quantitative PCR. Sli-miR-14 and sli-miR-2a could be detected successfully and their expression patterns showed similar characteristics with those in model insects, further suggesting stem-loop RT-PCR technology can be used for identification of highly conserved miRNAs in non-model insects. These results provide a simplified and efficient strategy for studying the structure and function of highly conserved miRNAs, especially some critical miRNAs in non-model insects.
Keywords: microRNA; precursor; identification; stem-loop RT-PCR; non-model insect; sequence conservation; Spodoptera litura
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Gao, L.; Zuo, H.; Liu, K.; Li, H.; Zhong, G. A New Strategy for Identification of Highly Conserved microRNAs in Non-Model Insect, Spodoptera litura. Int. J. Mol. Sci. 2012, 13, 612-627.
Gao L, Zuo H, Liu K, Li H, Zhong G. A New Strategy for Identification of Highly Conserved microRNAs in Non-Model Insect, Spodoptera litura. International Journal of Molecular Sciences. 2012; 13(1):612-627.
Gao, Lu; Zuo, Hongliang; Liu, Keling; Li, Haiyi; Zhong, Guohua. 2012. "A New Strategy for Identification of Highly Conserved microRNAs in Non-Model Insect, Spodoptera litura." Int. J. Mol. Sci. 13, no. 1: 612-627.