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Int. J. Mol. Sci. 2012, 13(1), 612-627; doi:10.3390/ijms13010612
Article

A New Strategy for Identification of Highly Conserved microRNAs in Non-Model Insect, Spodoptera litura

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Received: 19 October 2011; in revised form: 23 November 2011 / Accepted: 28 December 2011 / Published: 9 January 2012
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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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 microRNA; precursor; identification; stem-loop RT-PCR; non-model insect; sequence conservation; Spodoptera litura
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

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.

AMA Style

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.

Chicago/Turabian Style

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.



Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert