Identification of miRNAs in a Liver of a Human Fetus by a Modified Method

Background

miRNAs are 17–25 nucleotides long RNA molecules that have been found to regulate gene expression in human cells. There are studies showing that different groups of miRNAs are involved in development of different tissues. In hepatocytes there are reported particular types of miRNAs that regulate gene expression.

Methods

We established a human fetal liver cDNA library by a modified cloning protocol. Then plasmid isolation from the colonies was performed. After sequencing and database searching, the miRNAs were recognized. RT-PCR and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA.

Results

One novel miRNA was discovered, together with another 35 previously-known miRNAs in the fetal liver. Some of them existed in variants. The miRNAs identified were validated by RT-PCR and sequencing. Quantitative analysis showed that they have variable expression.

Conclusion

Our results indicate that a special group of miRNAs may play an important role in fetal liver development in a synergistic manner.     

Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus)

Background

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.

Methodology/Principal Findings

Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.

Conclusions/Significance

We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.     

Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Background

To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).

Methodology/Principal Findings

A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.

Conclusions

Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.     

Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

Objectives

The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing.

Methods and Materials

Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina’s mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression.

Results

Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12–20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17–20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5–6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20–25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and “novel” lincRNAs.

Conclusion

Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications.     

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

Background

Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.

Methodology and Results

We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.

Conclusions

Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.     

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

Background  

Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.     

Identification of chilling stress-responsive tomato microRNAs and their target genes by high-throughput sequencing and degradome analysis

Background

MicroRNAs (miRNAs) are a class of noncoding small RNAs (sRNAs) that are 20–24 nucleotides (nt) in length. Extensive studies have indicated that miRNAs play versatile roles in plants, functioning in processes such as growth, development and stress responses. Chilling is a common abiotic stress that seriously affects plants growth and development. Recently, chilling-responsive miRNAs have been detected in several plant species. However, little is known about the miRNAs in the model plant tomato. ‘LA1777’ (Solanum habrochaites) has been shown to survive chilling stress due to its various characteristics.

Results

Here, two small RNA libraries and two degradome libraries were produced from chilling-treated (CT) and non-chilling-treated (NT) leaves of S. habrochaites seedlings. Following high-throughput sequencing and filtering, 161 conserved and 236 novel miRNAs were identified in the two libraries. Of these miRNAs, 192 increased in the response to chilling stress while 205 decreased. Furthermore, the target genes of the miRNAs were predicted using a degradome sequencing approach. It was found that 62 target genes were cleaved by 42 conserved miRNAs, while nine target genes were cleaved by nine novel miRNAs. Additionally, nine miRNAs and six target genes were validated by quantitative real-time PCR (qRT-PCR). Target gene functional analysis showed that most target genes played positive roles in the chilling response, primarily by regulating the expression of anti-stress proteins, antioxidant enzyme and genes involved in cell wall formation.

Conclusions

Tomato is an important model plant for basic biological research. In this study, numerous conserved and novel miRNAs involved in the chilling response were identified using high-throughput sequencing, and the target genes were analyzed by degradome sequencing. The work helps identify chilling-responsive miRNAs in tomato and increases the number of identified miRNAs involved in chilling stress. Furthermore, the work provides a foundation for further study of the regulation of miRNAs in the plant response to chilling stress.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1130) contains supplementary material, which is available to authorized users.     

Evaluating bias-reducing protocols for RNA sequencing library preparation

Background

Next-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol).A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure.

Results

A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias.

Conclusions

The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-569) contains supplementary material, which is available to authorized users.     

Identification of Conserved and Novel microRNAs from Liriodendron chinense Floral Tissues

Background

Liriodendron chinense (L. chinense) is an endangered basal angiosperm plant in China because of its low reproductive efficiency. Recently, miRNAs have obtained great attention because they can play important roles. Through high throughput sequencing technique, large amount of miRNAs were identified from different plant species. But there were few studies about the miRNAs in the basal angiosperms especially in the sexual reproduction process.

Results

Deep sequencing technology was applied to discover miRNAs in L. chinense flowers at different stages. After bioinformatic analysis, 496 putative conserved miRNAs representing 97 families and 2 novel miRNAs were found. Among them, one is previously regarded as gymnosperm specific. Their expressions were further validated by Real-time PCR for 13 selected miRNAs. Putative targeting genes were predicted and categorized with gene ontology (GO) analysis. About ten percents of the targets are involved in the reproduction process. Further expressional analysis showed that many of these miRNAs were highly related to the reproductive growth.

Conclusions

This is the first comprehensive identification of conserved and novel miRNAs in L. chinense. The data presented here might not only help to fill the gap of miRNA registered about basal angiosperm plants but also contribute to understanding the evolution of miRNAs. The differential expression of some of the miRNAs and the prediction of their target genes are also helpful in understanding the regulation of L. chinense sexual reproduction.     

Identification of baboon microRNAs expressed in liver and lymphocytes

Background  

MicroRNAs (miRNAs) are small noncoding RNAs (~22 nucleotides) that regulate gene expression by cleaving mRNAs or inhibiting translation. The baboon is a well-characterized cardiovascular disease model; however, no baboon miRNAs have been identified. Evidence indicates that the baboon and human genomes are highly conserved; based on this conservation, we hypothesized that comparative genomic methods could be used to identify baboon miRNAs.