Solexa High-throughput Sequencing Research Articles

Genes Selectively Expressed in Rat Organs.

Understanding organic functions at a molecular level is important for scientists to unveil the disease mechanism and to develop diagnostic or therapeutic methods. The present study tried to find genes selectively expressed in 11 rat organs, including the adrenal gland, brain, colon, duodenum, heart, ileum, kidney, liver, lung, spleen, and stomach. Three normal male Sprague-Dawley (SD) rats were anesthetized, their organs mentioned above were harvested, and RNA in the fresh organs was extracted. Purified RNA was reversely transcribed and sequenced using the Solexa high-throughput sequencing technique. The abundance of a gene was measured by the expected value of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM). Genes in organs with the highest expression level were sought out and compared with their median value in organs. If a gene in the highest expressed organ was significantly different (p < 0.05) from that in the medianly expressed organ, accompanied by q value < 0.05, and accounted for more than 70% of the total abundance, the gene was assumed as the selective gene in the organ. The Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) pathways were enriched by the highest expressed genes. Based on the criterion, 1,406 selective genes were screened out, 1,283 of which were described in the gene bank and 123 of which were waiting to be described. KEGG and GO pathways in the organs were partly confirmed by the known understandings and a good portion of the pathways needed further investigation. The novel selective genes and organic functional pathways are useful for scientists to unveil the mechanisms of the organs at the molecular level, and the selective genes' products are candidate disease markers for organs.

Genetic analysis of DNA methylation in dyslipidemia: a case-control study.

Coronary heart disease has become the leading cause of death in developed countries, and dyslipidemia is closely associated with the risk of cardiovascular disease. Dyslipidemia is caused by the abnormal regulation of several genes and signaling pathways, and dyslipidemia is influenced mainly by genetic variation. AMFR, FBXW7, INSIG1, INSIG2, and MBTPS1 genes are associated with lipid metabolism. In a recent GWAS study, the GRINA gene has been reported to be associated with dyslipidemia, but its molecular mechanism has not been thoroughly investigated. The correlation between the DNA methylation of these genes and lipid metabolism has not been studied. This study aimed to examine the relationship between the DNA methylation of these genes and the risk of dyslipidemia by comparing the methylation levels of dyslipidemia and control samples. A case-control research method was used in this study. The patient's blood samples were collected at the Heart Center of the First Affiliated Hospital of Xinjiang Medical University. In the Xinjiang Han population, 100 cases of hyperlipidemia and 80 cases of the control group were selected. The two groups were age and gender-matched. Quantitative methylation analysis of CpG sites in the gene promoter regions of six genes was performed by Solexa high-throughput sequencing. The DNA methylation levels of 23 CpG sites in six genes were shown to be associated with hyperlipidemia, and a total of 20 DNA methylation haplotypes showed statistically significant differences between the two groups. When compared with the control group, the dyslipidemia group had significantly higher levels of methylation in the GRINA gene (2.68 vs 2.36, P = 0.04). Additionally, we also discovered a significant methylation haplotype of GRINA (P = 0.017). The findings of this study reveal that the DNA methylation of GRINA increases the risk for dyslipidemia in humans.

Differential microRNA expression profile of Trichinella spiralis larvae after exposure to the host small intestinal milieu

The process by which Trichinella spiralis muscle larvae are activated to infect the intestine after exposure to the host small intestinal milieu is crucial for the successful establishment of T. spiralis infection. However, the molecular mechanism underlying the invasion of intestinal epithelial cells by T. spiralis has not been elucidated. MicroRNAs are a class of small noncoding RNAs that participate in parasite growth and development, pathogenic processes, and host-parasite interactions. In the present study, the differential expression profile of miRNAs in T. spiralis after exposure to the mouse small intestinal milieu was analysed using Solexa high-throughput sequencing technology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to analyse the functions of miRNA target genes, and dual-luciferase reporter assays were subsequently applied to test the regulatory effects of one significantly decreased miRNA (let-7) on its four predicted target genes. In total, 2,000 known miRNAs (930 upregulated and 1070 downregulated) and 43 novel miRNAs (22 upregulated and 21 downregulated) were found to be differentially expressed in intestinal larvae, compared with muscle larvae. The KEGG pathway analysis showed that the predicted target genes of differentially expressed miRNAs were involved in 299 different pathways, and the top 10 pathways were metabolic pathways, biosynthesis of secondary metabolites, neuroactive ligand-receptor interaction, lysosome, focal adhesion, purine metabolism, starch and sucrose metabolism, tight junction, carbohydrate digestion and absorption, and pathways in cancer. As one of the most widely studied miRNA families, the expression of let-7 was significantly decreased in T. spiralis after exposure to host small intestinal milieu. A dual-luciferase reporter assay revealed that neuropeptide Y receptor type 6 and carboxypeptidase E were direct target genes of let-7, and were downregulated by binding with their 3′ UTR. GO function analysis showed that carboxypeptidase E had multiple enzymatic activities, suggesting that it might participate in cell membrane damage and larval invasion. These data suggest that the differentially expressed miRNAs in T. spiralis might have a regulatory role in the invasion of host intestinal epithelial cells. This study provides a new insight into the molecular mechanisms of invasion by T. spiralis and the regulatory functions of miRNAs in host-Trichinella interactions.

Screening candidate microR-15a- IRAK2 regulatory pairs for predicting the response to Staphylococcus aureus-induced mastitis in dairy cows.

We established a mastitis model using exogenous infection of the mammary gland of Chinese Holstein cows with Staphylococcus aureus and extracted total RNA from S. aureus-infected and healthy mammary quarters. Differential expression of genes due to mastitis was evaluated using Affymetrix technology and results revealed a total of 1230 differentially expressed mRNAs. A subset of affected genes was verified via Q-PCR and pathway analysis. In addition, Solexa high-throughput sequencing technology was used to analyze profiles of miRNA in infected and healthy quarters. These analyses revealed a total of 52 differentially expressed miRNAs. A subset of those results was verified via Q-PCR. Bioinformatics techniques were used to predict and analyze the correlations among differentially expressed miRNA and mRNA. Results revealed a total of 329 pairs of negatively associated miRNA/mRNA, with 31 upregulated pairs of mRNA and 298 downregulated pairs of mRNA. Differential expression of miR-15a and interleukin-1 receptor-associated kinase-like 2 (IRAK2), were evaluated by western blot and luciferase reporter assays. We conclude that miR-15a and miR-15a target genes (IRAK2) constitute potential miRNA-mRNA regulatory pairs for use as biomarkers to predict a mastitis response.

Functional Analysis of Three miRNAs in Agropyron mongolicum Keng under Drought Stress

Agropyron mongolicum Keng, a perennial diploid grass with high drought tolerance, belongs to the genus Agropyron, tribe Triticeae. It has made tremendous contributions toward reseeding natural pasture and seeding artificial grassland in China, especially in the arid and semi-arid area of northern China. As a wild relative of wheat, A. mongolicum is also a valuable resource for the genetic improvement of wheat crops. MicroRNAs are small non-coding RNA molecules ubiquitous in plants, which have been involved in responses to a wide variety of stresses including drought, salinity, chilling temperature. To date, little research has been done on drought-responsive miRNAs in A. mongolicum. In this study, two miRNA libraries of A. mongolicum under drought and normal conditions were constructed, and drought-responsive miRNAs were screened via Solexa high throughput sequencing and bioinformatic analysis. A total of 114 new miRNAs were identified in A. mongolicum including 53 conservative and 61 unconservative miRNAs, and 1393 target genes of 98 miRNAs were predicted. Seventeen miRNAs were found to be differentially expressed under drought stress, seven (amo-miR21, amo-miR62, amo-miR82, amo-miR5, amo-miR77, amo-miR44 and amo-miR17) of which were predicted to target on genes involved in drought tolerance. QRT-PCR analysis confirmed the expression changes of the seven drought related miRNAs in A. mongolicum. We then transformed the seven miRNAs into Arabidopsis thaliana plants, and three of them (amo-miR21, amo-miR5 and amo-miR62) were genetically stable. The three miRNAs demonstrated the same expression pattern in A. thaliana as that in A. mongolicum under drought stress. Findings from this study will better our understanding of the molecular mechanism of miRNAs in drought tolerance and promote molecular breeding of forage grass with improved adaption to drought.

A novel miRNA, miR-13664, targets CpCYP314A1 to regulate deltamethrin resistance in Culex pipiens pallens.

Extensive insecticide use has led to the resistance of mosquitoes to these insecticides, posing a major barrier to mosquito control. Previous Solexa high-throughput sequencing of Culex pipiens pallens in the laboratory has revealed that the abundance of a novel microRNA (miRNA), miR-13664, was higher in a deltamethrin-sensitive (DS) strain than a deltamethrin-resistant (DR) strain. Real-time quantitative PCR revealed that the miR-13664 transcript level was lower in the DR strain than in the DS strain. MiR-13664 oversupply in the DR strain increased the susceptibility of these mosquitoes to deltamethrin, whereas inhibition of miR-13664 made the DS strain more resistant to deltamethrin. Results of bioinformatic analysis, quantitative reverse-transcriptase polymerase chain reaction, luciferase assay and miR mimic/inhibitor microinjection revealed CpCYP314A1 to be a target of miR-13664. In addition, downregulation of CpCYP314A1 expression in the DR strain reduced the resistance of mosquitoes to deltamethrin. Taken together, our results indicate that miR-13664 could regulate deltamethrin resistance by interacting with CpCYP314A1, providing new insights into mosquito resistance mechanisms.

Methotrexate combined with methylprednisolone for the recovery of motor function and differential gene expression in rats with spinal cord injury.

Methylprednisolone is a commonly used drug for the treatment of spinal cord injury, but high doses of methylprednisolone can increase the incidence of infectious diseases. Methotrexate has anti-inflammatory activity and immunosuppressive effects, and can reduce inflammation after spinal cord injury. To analyze gene expression changes and the molecular mechanism of methotrexate combined with methylprednisolone in the treatment of spinal cord injury, a rat model of spinal cord contusion was prepared using the PinPoint™ precision cortical impactor technique. Rats were injected with methylprednisolone 30 mg/kg 30 minutes after injury, and then subcutaneously injected with 0.3 mg/kg methotrexate 1 day after injury, once a day, for 2 weeks. TreadScan gait analysis found that at 4 and 8 weeks after injury, methotrexate combined with methylprednisolone significantly improved hind limb swing time, stride time, minimum longitudinal deviation, instant speed, footprint area and regularity index. Solexa high-throughput sequencing was used to analyze differential gene expression. Compared with methylprednisolone alone, differential expression of 316 genes was detected in injured spinal cord treated with methotrexate and methylprednisolone. The 275 up-regulated genes were mainly related to nerve recovery, anti-oxidative, anti-inflammatory and anti-apoptotic functions, while 41 down-regulated genes were mainly related to proinflammatory and pro-apoptotic functions. These results indicate that methotrexate combined with methylprednisolone exhibited better effects on inhibiting the activity of inflammatory cytokines and enhancing antioxidant and anti-apoptotic effects and thereby produced stronger neuroprotective effects than methotrexate alone. The 316 differentially expressed genes play an important role in the above processes.

Gene Expression Profiling of Broiler Liver under Cold Stress by High-Throughput Sequencing Technology.

Cold stress is a major environmental factor restricting the sustainable development of animal husbandry. To gain insight into the gene-regulation processes in broilers under cold stress, gene expression profiling was conducted using high-throughput Solexa sequencing of broiler liver tissue under cold stress conditions and control conditions. According to Solexa sequencing, we identified 255 genes whose expression levels differed between the treatment and control group. Under cold stress, 135 genes were up-regulated and 120 genes were down-regulated genes compared with levels in the control group. Moreover, 469 genes were expressed only in the control group, and 172 genes were expressed only in the treatment group. These data were confirmed by real-time quantitative PCR. Gene Ontology enrichment analysis showed that the differentially expressed genes (DEGs) were mainly enriched in material metabolism and immune functions. KEGG enrichment analysis showed that DEGs were enriched in pyruvate metabolism, glycolysis/gluconeogenesis, fatty acid metabolism, insulin signaling pathway and others. In conclusion, these results may serve as an important reference for broiler breeding and provide new clues for the elucidation of molecular mechanisms of cold stress.

Comparative Analysis of the Bacterial Community on Anodic Biofilms in Sediment Microbial Fuel Cell Under Open and Closed Circuits

To investigate the differences in microbial community of anodic biofilms under open and closed circuits, sediment microbial fuel cell (SMFCs) reactors connecting with a 5 kΩ external resistance and open circuit during the start-up period were operated individually. Anodic biofilms were collected and analyzed using Solexa high-throughput sequencing technology. A total of 3936 and 3930 operational taxonomic units (OTUs) were obtained from the anodic biofilms under open and closed circuits, respectively. After 97% similarity merging, 1581 and 1551 OTUs were finally determined from open and close circuit biofilms, respectively. The analysis of α diversity showed that bacterial diversity of anodic biofilm under open circuit was higher than that under closed circuit. The dominant bacterial were Proteobacteria, Firmicutes and Bacteroidetes for both open and closed circuits. They accounted for 59.79%, 12.54% and 9.02% under open circuit biofilm respectively; and these values were 63.02%, 10.01% and 3.60% in the closed circuit biofilm respectively, and Geobacter accounted for 16.55% in the closed circuit biofilm. The present study demonstrated that the electron transfer process during start-up period affected the microbial community structure of the anodic biofilms.

Identification of microRNAs regulating Escherichia coli F18 infection in Meishan weaned piglets.

Background Escherichia coli F18 is mainly responsible for post-weaning diarrhea (PWD) in piglets. The molecular regulation of E. coli F18 resistance in Chinese domestic weaned piglets is still obscure. We used Meishan piglets as model animals to test their susceptibility to E. coli F18. Small RNA duodenal libraries were constructed for E. coli F18-sensitive and -resistant weaned piglets challenged with E. coli F18 and sequenced using Illumina Solexa high-throughput sequencing technology.ResultsSequencing results showed that 3,475,231 and 37,198,259 clean reads were obtained, with 311 known miRNAs differently expressed in resistant and sensitive groups, respectively. Twenty-four miRNAs, including 15 up-regulated and 9 down-regulated, demonstrated more than a 2-fold differential expression between the F18-resistant and -sensitive piglets. Stem-loop RT-qPCR showed that miR-136, miR-196b, miR-499-5p and miR-218-3p significantly expressed in intestinal tissue (p < 0.05). KEGG pathway analysis for target genes revealed that differently expressed miRNAs were involved in infectious diseases, signal transduction and immune system pathways. Interestingly, the expression of miR-218-3p in intestinal tissue had a very significant negative correlation with target DLG5 (P < 0.01).ConclusionsBased on the expression correlation between miRNA and target genes analysis, we speculate that miR-218-3p targeting to DLG5, appears to be very promising candidate for miRNAs involved in response to E. coli F18 infection. The present study provides improved database information on pig miRNAs, better understanding of the genetic basis of E. coli F18 resistance in local Chinese pig breeds and lays a new foundation for identifying novel markers of E. coli F18 resistance.ReviewersThis article was reviewed by Neil R Smalheiser and Weixiong Zhang.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0160-3) contains supplementary material, which is available to authorized users.

MicroRNA-155 is a potential molecular marker of natural killer/T-cell lymphoma

Natural killer/T-cell lymphoma (NKTCL) is characterized by its highly aggressive nature and rapid progression. MicroRNAs (miRNAs) play key roles in the development of NKTCL. We utilized next-generation Solexa high-throughput sequencing to compare miRNA expression in the SNK-6 and YTS NKTCL cell lines with expression in normal NK cells. We found that 195 miRNAs were upregulated in the SNK-6 cells and 286 miRNAs were upregulated in the YTS cells. Based on those results, we selected six miRNAs, including miRNA-155, and confirmed their expression using real-time polymerase chain reaction. Expression of miRNA-155 was higher in SNK-6 and YKS cells than in normal NK cells. We next determined the levels of miRNA-155 in the serum of healthy individuals and NKTCL patients, and correlated its expression with clinical parameters and inflammatory factors detected using enzyme-linked immunoabsorbent assays. Levels of miRNA-155 were higher in NKTCL patients’ serum than in serum from healthy individuals. miRNA-155 expression was higher in patients with stable or progressive disease (SD+PD) than in those with partial or complete remission (PR+CR). While further studies are needed to clarify the underlying molecular mechanisms, it appears miRNA-155 may be a molecular marker of NKTCL.

Influence of Carbonization Temperature on Bacterial Community of the Biological Carbon Electrode Based on High-throughput Sequencing Technology

Microbial diversity of anodic biofilm in bioelectrochemical systems with hemp rod carbonized at 1000 and 1800℃ as anode was investigated using Solexa high-throughput sequencing technology. The results showed that a total of 4231 and 5263 optimized 16S rRNA gene sequences were gained from the electrode biofilm on the hemp rod carbonized at 1000 and 1800℃, respectively. At the level of 97% similarity, 1187 and 1338 OTUs were obtained for electrode biofilm carbonized at 1000 and 1800℃, respectively. The result of α diversity analysis showed that microbial diversity increased with decreasing carbonization temperature. Dominant phylum for both biofilms were Proteobacteria, Firmicutes and Bacteroidetes, which accounted for 66%, 10% and 9%, respectively for 1000℃, while 71%, 7% and 9%, respectively for 1800℃. Beside the coexisting phylum, some unique species were also discovered, demonstrating that carbonization temperature did not only influence the electrode structure, but also affected the microbial community structure.

Gene screening and differential expression analysis of microRNAs in the middle silk gland of wild-type and naked pupa mutant silkworms (Bombyx mori)

MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that serve as important post-transcriptional gene expression regulators and play important roles in the silkworm (Bombyx mori) development, growth, and viral immunity. However, information on the diversity of these regulatory RNAs in the middle silk gland (MSG) of naked pupa (Nd) mutant silkworms is limited. In this study, by using Solexa high-throughput sequencing technology, we identified and compared small RNA libraries from the MSG of wild-type silkworm P50(MSG-P50) and the Nd mutant (MSG-Nd), respectively. A total of 272 conserved and 333 novel miRNAs were identified, in which 141 ones showed significantly different expression patterns between MSG-P50 and MSG-Nd, and 10 ones were randomly selected and validated by stem-loop quantitative reverse-transcription polymerase chain reaction (qRT-PCR). In addition, potential targets were predicted for differentially expressed miRNAs based on sequence complementation between miRNAs and their target genes. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed miRNAs that actively participate in various life processes and three pathways associated with protein synthesis including endoplasmic reticulum pathway, ribosome pathway, and ribosome biogenesis in eukaryotes, were significantly disrupted in MSG-Nd. This is the first comprehensive description of miRNAs in the silkworm MSG. Overall, the results provide useful information for future studies on miRNAs and suggest that the fibroin synthetic deficiency in the posterior silk gland impairs the sericin secretion process in MSG.

Complete genome sequence and comparative genome analysis of the Paenibacillus mucilaginosus K02

AimPaenibacillus mucilaginosus (P. mucilaginosus) K02 is implicated in mineral weathering. However, relevant molecular mechanisms remain obscure. The study aims to uncover the bacterium's physiological processes using genomic approaches. Methods and resultsGenomic DNA from P. mucilaginosus K02 was sequenced using high-throughput Solexa sequencing technology and then conducted for Clusters of Orthologous Group (COG) annotation. Thereafter, genome sequences of K02 were compared with two strains, 3016 and KNP414. Mummer was applied for collinearity analysis of three P. mucilaginosus genomes. BLAST was used to identify pan and core genes in these strains. Finally, a phylogenetic tree was constructed using the maximum likelihood method by TreeBeST. Complete genome sequence of P. mucilaginosus K02 indicated the strain comprises one circular chromosome with 8,819,200 bases containing 58.3% GC content and 84.75% coding regions. A total of 7299 predicted ORFs were identified in the genome, among them, several genes were related to carbonic anhydrase (CA), and exopolysaccharide biosynthesis and secretion. Moreover, proteins of the predicted genes were annotated in COG categories such as “Carbohydrate transport and metabolism” and “Inorganic ion transport and metabolism.” In comparison with KNP414 and 3016, K02 exhibited chromosomal recombination or transposition. A total of 6662 core genes were identified among three P. mucilaginosus strains. The phylogenomic study indicated that P. mucilaginosus K02 was clustered with P. mucilaginosus strains 3016 and KNP414. ConclusionsIn P. mucilaginosus K02, genes related to CA and exopolysaccharide biosynthesis and secretion, and that involved in metabolism-related processes might play significant roles in mineral weathering.

Identification of novel and differentially expressed microRNAs in ovine ovary and testis tissues using Solexa sequencing and bioinformatics

MicroRNAs (miRNAs) are small, single stranded, non-coding RNA molecules, about 19–25 nucleotides in length, which regulate the development and functions of reproductive system of mammal. To discover novel miRNAs and identify the differential expression of them in ovine ovary and testis tissues, the study constructed two libraries by using next-generation sequencing technologies (Solexa high-throughput sequencing technique). As a result, 9 321 775 and 9 511 538 clean reads were obtained from the ovary and testis separately, which included 130 562 (90 genes of ovary) and 56 272 (85 genes of testis) of known miRNAs and 486 potential novel miRNAs reads. In this study, a total of 65 conserved miRNAs were significantly differentially expressed (P<0.01) between the two samples. Among them, 28 miRNAs were up-regulated and 3 miRNAs were down-regulated on ovary compared with testis. In addition, the known miRNAs with the highest expression level (5 miRNAs) and 30 novel miRNAs with the functions related to reproduction were validated using the real-time quantitative RT-PCR. Moreover, the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that differentially expressed miRNAs were involved in ovary and testis physiology, including signal transduction, gonad development, sex differentiation, gematogenesis, fertilization and embryo development. The results will be helpful to facilitate studies on the regulation of miRNAs during ruminant reproduction.

PD23-07 A PROSPECTIVE TRANSLATIONAL STUDY OF MICRORNA-519A DETECTION AS A URINE MARKER IN OUTPATIENTS WITH MACROSCOPIC HEMATURIA: A PROMISING CANDIDATE MICRORNA FROM DEEP SEQUENCING SIGNATURE OF UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Detection and Screening I1 Apr 2015PD23-07 A PROSPECTIVE TRANSLATIONAL STUDY OF MICRORNA-519A DETECTION AS A URINE MARKER IN OUTPATIENTS WITH MACROSCOPIC HEMATURIA: A PROMISING CANDIDATE MICRORNA FROM DEEP SEQUENCING SIGNATURE OF UROTHELIAL CARCINOMA Hideki Enokida, Yasutoshi Yamada, Satoru Inoguchi, Tomoaki Ishihara, Shuichi Tatarano, Toshihiko Itesako, Naohiko Seki, and Masayuki Nakagawa Hideki EnokidaHideki Enokida More articles by this author , Yasutoshi YamadaYasutoshi Yamada More articles by this author , Satoru InoguchiSatoru Inoguchi More articles by this author , Tomoaki IshiharaTomoaki Ishihara More articles by this author , Shuichi TataranoShuichi Tatarano More articles by this author , Toshihiko ItesakoToshihiko Itesako More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1423AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Currently, tumor marker for bladder cancer (BC) depends on urine cytology because of its high specificity despite of its low sensitivity. MicroRNAs (miRNAs) are small noncoding RNAs of about 22 nucleotides in length. Because of its short structure, miRNAs are resistant to degradation and robustly detected in bodily fluids. At past AUA meeting, we have demonstrated that detection of several miRNAs in urine was useful biomarkers from retrospective cohort of UC patients. In this study, we performed our translational prospective study for miRNAs detection in urine as a diagnostic marker for detecting UC by using a new candidate miRNA based on deep sequencing miRNA analysis. METHODS Urine samples were randomly collected from 262 outpatients, who complained of macroscopic hematuria in our affiliate hospitals between November, 2011 and November, 2013. A total of 10 ml urine was centrifuged at 2000 g for 15 min. Total RNA of urine was extracted from both urine sediment and 5 ml of supernatant. We detected differentially expressed miRNAs in 5 BCs by using the next generation high through-put Solexa sequencing technology. A real-time PCR based experiment was used to evaluate expression levels of the miRNAs. On the basis of absolute quantification method, the expression level of each miRNA was calculated by a following formula: expression level = 2^(40-Ct). RESULTS Finally, urothelial carcinoma were pathologically proven in 78 patients (70 BCs and 8 renal pelvic cancers); urinary tract infection was found in 80 patients; urinary stone was in 46; and others was in 58. In urine sediment, the expression levels of miR-519a in UCs were significantly higher than that in non-UCs (UTIs, stones, and others) (P<0.0001). There was a significance that a higher expression levels of miR-519a was associated with high grade UCs compared with low grade UCs (P=0.0018). The ROC curve analysis revealed that miR-519a has good sensitivity of 62.8% and specificity of 76.2% to distinguish UCs from non-UCs. miR-519a was able to be detected in 26 of 45 patients who had negative urine cytology test. In terms of urine supernatant, we also found a significant difference of miR-519a expression level between UCs and non-UCs (P<0.0001). However, there was a relatively lower sensitivity of 52.2% and specificity of 78.2% to distinguish UCs from non-UCs. CONCLUSIONS Our translational study suggests that detection of miR-519a in urine may serve as potential diagnostic markers for UC detection. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e483 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hideki Enokida More articles by this author Yasutoshi Yamada More articles by this author Satoru Inoguchi More articles by this author Tomoaki Ishihara More articles by this author Shuichi Tatarano More articles by this author Toshihiko Itesako More articles by this author Naohiko Seki More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression profiling of cadmium response genes in ramie (Boehmeria nivea L.) root.

Ramie (Boehmeria nivea), a perennial herb belongs to Urticaceae family, is a rapid growth and high biomass crop with highly tolerant and accumulative to heavy metals. However, the gene expression and regulation caused by cadmium (Cd) in ramie has not been well studied. In the present study, a gene expression database of ramie root in the absence (control) or presence of 100 μM Cd was established. Solexa high-throughput sequencing technology showed that 3,654,395 and 3,572,333 tags have been obtained from control and Cd treatment respectively. In total, 3887 genes were detected with significant differential expression levels, in which 2883 genes were up-regulated and 1004 genes were down-regulated. Gene ontology and pathway-based analyses were performed to determine and further to understand the biological functions of those differentially expressed genes. Fifteen genes were selected and their expression levels were confirmed by quantitative RT-PCR, and twelve of them showed consistent expression patterns with the digital gene expression data. Results on these expression profiling of genes lay the basis for biotechnological modification of new transgenic plants with improved phytoremediation capacity.

Transcriptome profiling of root microRNAs reveals novel insights into taproot thickening in radish (Raphanus sativus L.).

BackgroundRadish (Raphanus sativus L.) is an economically important root vegetable crop, and the taproot-thickening process is the most critical period for the final productivity and quality formation. MicroRNAs (miRNAs) are a family of non-coding small RNAs that play an important regulatory function in plant growth and development. However, the characterization of miRNAs and their roles in regulating radish taproot growth and thickening remain largely unexplored. A Solexa high-throughput sequencing technology was used to identify key miRNAs involved in taproot thickening in radish.ResultsThree small RNA libraries from ‘NAU-YH’ taproot collected at pre-cortex splitting stage, cortex splitting stage and expanding stage were constructed. In all, 175 known and 107 potential novel miRNAs were discovered, from which 85 known and 13 novel miRNAs were found to be significantly differentially expressed during taproot thickening. Furthermore, totally 191 target genes were identified for the differentially expressed miRNAs. These target genes were annotated as transcription factors and other functional proteins, which were involved in various biological functions including plant growth and development, metabolism, cell organization and biogenesis, signal sensing and transduction, and plant defense response. RT-qPCR analysis validated miRNA expression patterns for five miRNAs and their corresponding target genes.ConclusionsThe small RNA populations of radish taproot at different thickening stages were firstly identified by Solexa sequencing. Totally 98 differentially expressed miRNAs identified from three taproot libraries might play important regulatory roles in taproot thickening. Their targets encoding transcription factors and other functional proteins including NF-YA2, ILR1, bHLH74, XTH16, CEL41 and EXPA9 were involved in radish taproot thickening. These results could provide new insights into the regulatory roles of miRNAs during the taproot thickening and facilitate genetic improvement of taproot in radish.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0427-3) contains supplementary material, which is available to authorized users.

Identification of a novel miRNA from the ovine ovary by a combinatorial approach of bioinformatics and experiments.

MicroRNAs (miRNAs) are a class of short endogenous, single-stranded, non-coding small RNA molecules, about 19–25 nucleotides in length that regulate gene expression at the translation level and influence many physiological process, such apoptosis, metabolism, signal transduction, and occurrence and development of diseases. In this study, we constructed a library from the ovine luteal phase ovary by using next-generation sequencing technology (Solexa high-throughput sequencing technique) and identified 267 novel miRNAs by bioinformatics. One of the novel miRNAs (ovis_aries_ovary-m0033_3p), which expressed in the sheep ovary and testis, was confirmed by real time PCR and northern blot. Ovis_aries_ovary-m0033_3p was 21 nucleotides in length and located on chromosome 12, and it had 100% similarity to hsa-miR-214-3p, mmu-miR-214-3p, dre-miR-214and ssc-miR-214. Meanwhile, the pre-miRNA was 82 nucleotides in length and had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_ovary-m0033_3p had a function similar to hsa-miR-214-3p, which is involved in the fine regulation of cell survival, embryonic development, breeding activities and resistance to ovarian cancer, so we defined it as oar-miR-214-3p. These experimental results will enrich the miRNA database for ovis aries and provide the basis for researching the regulation mechanism of miRNA in relation to breeding activities of seasonal breeding animals.

Identification and characterization of microRNAs related to salt stress in broccoli, using high-throughput sequencing and bioinformatics analysis.

BackgroundMicroRNAs (miRNAs) are a new class of endogenous regulators of a broad range of physiological processes, which act by regulating gene expression post-transcriptionally. The brassica vegetable, broccoli (Brassica oleracea var. italica), is very popular with a wide range of consumers, but environmental stresses such as salinity are a problem worldwide in restricting its growth and yield. Little is known about the role of miRNAs in the response of broccoli to salt stress. In this study, broccoli subjected to salt stress and broccoli grown under control conditions were analyzed by high-throughput sequencing. Differential miRNA expression was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). The prediction of miRNA targets was undertaken using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) database and Gene Ontology (GO)-enrichment analyses.ResultsTwo libraries of small (or short) RNAs (sRNAs) were constructed and sequenced by high-throughput Solexa sequencing. A total of 24,511,963 and 21,034,728 clean reads, representing 9,861,236 (40.23%) and 8,574,665 (40.76%) unique reads, were obtained for control and salt-stressed broccoli, respectively. Furthermore, 42 putative known and 39 putative candidate miRNAs that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time RT-PCR. Amongst these, the putative conserved miRNAs, miR393 and miR855, and two putative candidate miRNAs, miR3 and miR34, were the most strongly down-regulated when broccoli was salt-stressed, whereas the putative conserved miRNA, miR396a, and the putative candidate miRNA, miR37, were the most up-regulated. Finally, analysis of the predicted gene targets of miRNAs using the GO and KO databases indicated that a range of metabolic and other cellular functions known to be associated with salt stress were up-regulated in broccoli treated with salt.ConclusionA comprehensive study of broccoli miRNA in relation to salt stress has been performed. We report significant data on the miRNA profile of broccoli that will underpin further studies on stress responses in broccoli and related species. The differential regulation of miRNAs between control and salt-stressed broccoli indicates that miRNAs play an integral role in the regulation of responses to salt stress.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0226-2) contains supplementary material, which is available to authorized users.