MicroRNA Discovery and Validation in Silkworm – Case Study

The study of microRNA (miRNA) is growing rapidly as researchers discover new miRNA sequences and uncover the importance of these small regulatory elements linked to a wide range of biological functions. The miRBase sequence database (1) is the primary public repository for newly discovered miRNAs and the number of miRBase entries has grown rapidly from a mere 218 in 2002 to almost 10,000 in the latest version, suggesting the existence of many more miRNAs yet to be discovered.
Contributing to the rapid rate of new discoveries is the development of several new advanced technologies such as high-throughput sequencing and custom microfluidic arrays. The increasing availability of these technologies makes the discovery of new sequences in lesser understood organisms now routinely possible. A straightforward process of discovery, confirmation, and validation is commonly employed:

Deep Sequencing – Discovery —> Microarray – Confirmation —> QPCR – Validation

In a recent study, many novel miRNAs were experimentally verified in the silkworm Bombyx mori, an industrially important insect, by researchers at the Chinese Academy of Sciences, Shanghai using this three step process (2).
Deep sequencing (Illumina GA sequencing-by-synthesis platform) revealed a total of 95,184 non-redundant tags that matched to the silkworm genome, and computational pipeline analysis identified 3,750 potential miRNA precursors. Probes for these candidate miRNAs in situ synthesized on a custom microfluidic microarray (3) (LC Sciences µParaflo® microfluidic platform) confirmed 354 of the candidates. Further microarray experiments profiled the expression level of these miRNAs at the various developmental stages of the silkworm (egg, larvae, pupa, and adult). QPCR (GenePharma, molecular beacon platform) validated several highly differentially expressed miRNAs, thought to be important for embryogenesis and metamorphosis.
It is well known that miRNAs are important players for regulation of cellular activities. Therefore, the establishment of miRNAs in traditional industrial or agriculturally important species such as the silkworm should lead to better understanding of the fundamentals of their growth, maturation, and disease-resistance.

1. The miRBase sequence database is a comprehensive database of miRNA sequence data, annotation, and predicted gene targets and is the primary public repository for these data. miRBase also provides a gene-naming service for assigning official miRNA names to novel miRNAs before they are published. It is freely available to all at http://microrna.sanger.ac.uk/.

2. Zhang Y, Zhou X, Ge X, Jiang J, Li M, Jia S, Yang X, Kan Y, Miao X, Zhao G, Li F, Huang Y. (2009) Insect-Specific microRNA Involved in the Development of the Silkworm Bombyx mori. PLoS ONE 4(3), e4677.

3. More information about LC Sciences miRNA discovery and analysis services is available at: http://www.lcsciences.com/mirna_discovery.html.