Enrichment and verification of differentially expressed miRNAs in the ovarian tissue of Leizhou black ducks

Background Ovary is an important reproductive organ for poultry. MicroRNA (miRNA) is a highly conserved class of small non-coding RNA that function in a specific manner to post-transcriptionally regulate gene expression in organisms. Currently, miRNA has been studied extensively but research on duck ovarian tissue is relatively rare. Thus, in this study, we performed the first miRNA analysis of ovarian tissues in Leizhou black ducks with low and high rates of egg production. Method: Using high-throughput sequencing technology, miRNA library was constructed to obtain miRNA expression profile; differentially expressed miRNAs were screened, to predict miRNA target genes. Results A total of 29 differentially expressed miRNAs were obtained from the miRNA library, of which 7 were up-regulated and 22 were down-regulated. Verification of 12 randomly selected miRNAs, using RT-qPCR technology showed the results are consistent with the sequencing data, indicating that the sequencing results are reliable. The GO enrichment and KEGG pathway enrichment analysis of differential miRNAs showed that target genes were significantly enriched in signal pathways related to ovarian development, such as the oxytocin signaling pathway, GnRH signaling pathway, progesterone-mediated oocyte maturation, and AMPK signal pathway. Conclusion The research results enrich the data resources of duck miRNAs, and provide a theoretical basis for further research on the laying performance of poultry and molecular-assisted breeding of poultry in the future.

An extension to: Systematic assessment of commercially available low-input miRNA library preparation kits

High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. To assist researchers choose the most appropriate library preparation kit, we recently compared the performance of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. As before, performance was assessed with respect to sensitivity, reliability, titration response and differential expression. Despite NEXTflex employing partially-randomised adapter sequences to minimise bias, we reaffirm that biases in miRNA abundance are kit-specific, complicating the comparison of miRNA datasets generated using different kits.

ILLUMINA®TRUSEQ® VS NEBNEXT® SMALL RNA LIBRARY PREPARATION KIT FOR MIRNA PROFILING IN PERSICARIA MINOR: WHICH BETTER?

In plants, a group of non-coding small RNA (sRNA) has been provento be an important player in regulating gene expression that can govern network of genetic systems. The two major classes of sRNA which are very extensively studied through deep sequencing, microRNA (miRNA) and small-interfering RNA (siRNA) classes, are well documented. However, the isolation method of sRNA differs depending on the type of sample. Here, we demonstrate the miRNA library preparation using two different Small RNA Library preparation kit, Illumina®TruSeq® Small RNA Preparation and NEBNext® Multiplex Small RNA Library Preparation kit on a plant rich in secondary metabolite Persicaria minor using recommended protocol. The result show NEBNext® Multiplex Small RNA Library Preparation kit can recover small RNA better than Illumina®TruSeq® Small RNA Preparation kit. Thus, this study recommended NEBNext® Multiplex Small RNA Library Preparation kit for miRNA library preparation on Persicaria minor.

mRNA/microRNA Profile at the Metamorphic Stage of Olive Flounder (Paralichthys olivaceus)

Flatfish is famous for the asymmetric transformation during metamorphosis. The molecular mechanism behind the asymmetric development has been speculated over a century and is still not well understood. To date, none of the metamorphosis-related genes has been identified in flatfish. As the first step to screen metamorphosis-related gene, we constructed a whole-body cDNA library and a whole-body miRNA library in this study and identified 1051 unique ESTs, 23 unique miRNAs, and 4 snoRNAs in premetamorphosing and prometamorphosingParalichthys olivaceus. 1005 of the ESTs were novel, suggesting that there was a special gene expression profile at metamorphic stage. Four miRNAs (pol-miR-20c,pol-miR-23c,pol-miR-130d, andpol-miR-181e) were novel toP. olivaceus; they were characterized as highly preserved homologies of published miRNAs but with at least one nucleotide differed. Representative 24 mRNAs and 23 miRNAs were quantified during metamorphosis ofP. olivaceusby using quantitative RT PCR or stem-loop qRT PCR. Our results showed that 20 of mRNAs might be associated with early metamorphic events, 10 of mRNAs might be related with later metamorphic events, and 16 of miRNAs might be involved in the regulation of metamorphosis. The data provided in this study would be helpful for further identifying metamorphosis-related gene inP. olivaceus.