Pooled RNA Samples Research Articles

204P - Analytical validation of OncoMasTR, a multigene test for predicting risk of distant recurrence in hormone receptor-positive early stage breast cancer

Background: OncoMasTR is a new multigene prognostic test that was discovered via a novel transcriptional network analysis method that identified upstream Master Transcription Regulators (MTRs), which regulate previously identified prognostic biomarkers. The optimised OncoMasTR signature incorporating clinicopathological information has been shown to be significantly prognostic for predicting distant recurrence in two independent cohorts (TransATAC & a subset of TAILORx from participating Irish centres). The analytical performance characteristics of the OncoMasTR test, comprising solely three prognostic MTRs, were determined. Methods: Relative gene expression levels were measured by RT-qPCR. Assay precision and input ranges were determined using a panel of samples representative of low and high recurrence risk tested across a number of runs incorporating different sources of variation. Serial dilutions of a pooled patient RNA sample was used to establish the linear range and efficiency of the individual gene assays. Results: The overall standard deviation of the OncoMasTR risk score was 0.15, which represents less than 2% of the 10-unit risk score range. The majority of the variability in OncoMasTR results was related to within-run variation (78.2%) with other between-run variation sources contributing relatively little (PCR instrument (0.6%), assay operator (5.2%), reagent lots (7.3%) or loading position (8.7%)). Consistent risk scores were measured for individual samples from 40ng down to 500-fold RNA input range corresponding to CT values of 23 – 36, demonstrating the ability of the test to reliably detect low level expression of the OncoMasTR panel. Importantly, PCR efficiencies were similar for the individual MTR and reference gene assays which ranged from 79 – 95%. Conclusions: The OncoMasTR prognostic test displays robust analytical and clinical performance and is being launched as a CE-marked test. The concise nature of the three gene signature and a simplified workflow that can be readily adopted using standard laboratory equipment will enable convenient qualification by local laboratories for decentralised use. (Less)

103. Dysregulated circulating microRNAs in preeclampsia: The role of miR-574-5p and miR-1972 in endothelial dysfunction

MicroRNAs are small non-coding RNAs responsible for post-transcriptional regulation of gene expression. MicroRNAs might be involved in the pathogenesis of endothelial cell dysfunction of preeclampsia. Compare circulating microRNA concentrations in early-onset preeclampsia with healthy pregnancy. Subsequently determine if the differentially expressed microRNAs contribute to endothelial cell dysfunction in vitro. Total RNA was isolated from plasma EDTA samples of early-onset preeclamptic patients (n = 10) and gestational age-matched healthy pregnant women (n = 10). MicroRNA expression was measured by miRNA 3.1 arrays (Affymetrix) and validated by RT-RT-PCR. Subsequently, human umbilical vein endothelial cells (HUVEC) were transfected (lipofectamine RNAiMAX), with microRNA mimics (Invitrogen) of the 3 most significantly upregulated microRNAs in preeclampsia. Tube formation, wound healing, and proliferation assays were performed to study endothelial cell function. Microarray (Affymetrix) on pooled RNA samples of transfected HUVEC was done and validated by RT-RT-PCR. 41 microRNAs were differentially expressed (p <0.01, fold change >1.2 or <-1.2) in preeclamptic women compared to healthy pregnant women. The top 3 upregulated microRNAs were miR-1972, miR-574-5p and miR-4792-3p; the top 3 downregulated microRNAs were miR-548a-3p, miR-874 and miR-451. Transfection of HUVEC with the miR-574-5p mimic decreased the wound healing capacity after 12 h (66%), and reduced proliferation of HUVEC (32%); transfection with the miR-1972 mimic reduced the tube formation capacity (number of loops formed decreased 19%). Using these assays no effects of the miR-4793-3p mimic on endothelial cell function were found. Array and RT-RT-PCR data revealed that the miR-574-5p mimic significantly decreased the expression of the proliferation marker MKI67. Differentially expressed microRNAs during preeclampsia might affect endothelial cell function. The negative effects of the microRNA mimics on proliferation and tube formation might indicate decreased proliferation and angiogenesis capacity of endothelial cells during preeclampsia. These microRNAs thus might be key modulators of endothelial dysfunction during preeclampsia.

Abstract 5393: Exosomal microRNA profiles in peritoneal fluids as a therapeutic biomarker for peritoneal metastasis of gastric cancer

Abstract Background: Repeated intraperitoneal chemotherapy (IPC) using taxane is highly effective for peritoneal metastasis. Peritoneal fluids are supposed to contain various factors derived from tumor cells seeded in peritoneal cavity. This study aimed to explore possible biomarkers for suitable IPC using exosomal miRNA profiles derived from malignant ascites or peritoneal lavages. Methods: Peritoneal fluid samples were obtained from 11 patients with peritoneal metastasis (PM+) and 14 patients who received gastrectomy for early gastric cancer (PM-). Exosomal fractions were isolated using ultracentrifuge method and total RNAs were extracted. The pooled RNA samples in both groups were mixed and expression of miRNAs were analyzed using miRNome miScript miRNA PCR Array (QIAGEN). Based on these results, we constructed the custom miRNA PCR panel and evaluates expression of miRNAs in individual samples. Results: The comprehensive PCR analysis showed that total of 56 and 54 miRNAs were strongly expressed in exosomes in PM+ and PM- peritoneal fluid sample, respectively. In analysis of individual samples, 7 upregulated miRNAs (miR-150-5p, miR-223-3p, miR-204-5p, miR-720, and miR-92a, miR-21-5p, miR-4301, and miR-342-3p) and 3 downregulated miRNAs (miR-29a-3p, miR-29b-3p, and miR-29c-3p) were identified in PM+ samples. Conclusion: In patients who receive IPC, peritoneal fluids can be repeatedly obtained from peritoneal access port. Exosomal miRNA profiles of peritoneal fluid samples might be a useful biomarker to determine the therapeutic efficacy as well as to predict the outcome in those patients with peritoneal metastasis. Citation Format: Hideyuki Ohzawa, Yuko Kumagai, Hironori Yamaguchi, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Exosomal microRNA profiles in peritoneal fluids as a therapeutic biomarker for peritoneal metastasis of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5393.

Discovery and characterization of conserved and novel microRNAs from blunt snout bream (Megalobrama amblycephala) by deep sequencing

MicroRNAs (miRNAs) are short, single stranded RNA molecules with approximately 22 nts in length, which regulate the stability and translation of messenger RNAs in several organisms. To increase the repertoire of miRNAs characterized in M. amblycephala, we used the deep sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the 4 different tissues of M. amblycephala. A total of 309 conserved miRNAs that originated from 131 miRNA families were detected. 15 novel candidates miRNA were identified. Randomly selected 6 miRNAs were analyzed by stem-loop qRT-PCR and differential expression patterns were observed in 6 different tissues of M. amblycephala. Furthermore, the potential targets were predicted. GO analysis showed that most of the targets were involved in a broad range of physiological functions including fish growth, development, metabolism, stress responses and so on. Overall, our results significantly increased the number of novel miRNAs in M. amblycephala, which should be useful for further investigation into the role of miRNAs in regulating diverse biological processes.

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing

BackgroundDespite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms.ResultsThe sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes.ConclusionsThe transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.

De novo transcriptomic analysis to reveal functional genes involved in triterpenoid saponin biosynthesis in Oplopanax elatus NAKAI

Oplopanax elatus is a valuable medicinal plant in the family Araliaceae that recommends itself as source of herbal preparations. Although, as in ginseng, triterpenoid saponins make up the major bioactive component of O. elatus , nothing is known about the genes that are involved in the biosynthesis of these complex compounds, as reflected also by a lack of genomic information in public databases. Using Illumina paired-end sequencing technology, we have therefore generated a transcriptome library of O. elatus from a pooled RNA sample from different organs. 208,959 unigenes were assembled from approximately 77 million high-quality reads, and 110,202 unigenes (52.7% of the unigenes) were annotated. In addition, 47,273 cDNA-derived SSRs in 38,446 unigenes were identified as potential molecular markers. Furthermore, 122 unigenes encoding 47 putative enzymes related to the biosynthesis of the backbone of triterpenoid saponins were identified by analyzing our library. The organ-specific expression of selected genes suggests that the leaves of O. elatus are the main site of triterpenoid saponin biosynthesis. The transcriptome data reported here provides valuable and comprehensive information for further research into the metabolic pathways of O. elatus as well as into genetic variation in the Oplopanax genus. The online version of this article (doi: 10.5073/JABFQ.2017.090.004) contains supplementary files .

Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing.

BackgroundMicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not been explored.ResultsPomegranate, which accumulates a large amount of anthocyanins in skin and arils, is valuable to human health, mainly because of its antioxidant properties. In this study, we developed a small RNA library from pooled RNA samples from young seedlings to mature fruits and identified both conserved and pomegranate-specific miRNA from 29,948,480 high-quality reads. For the pool of 15- to 30-nt small RNAs, ~50 % were 24 nt. The miR157 family was the most abundant, followed by miR156, miR166, and miR168, with variants within each family. The base bias at the first position from the 5’ end had a strong preference for U for most 18- to 26-nt sRNAs but a preference for A for 18-nt sRNAs. In addition, for all 24-nt sRNAs, the nucleotide U was preferred (97 %) in the first position. Stem-loop RT-qPCR was used to validate the expression of the predominant miRNAs and novel miRNAs in leaves, male and female flowers, and multiple fruit developmental stages; miR156, miR156a, miR159a, miR159b, and miR319b were upregulated during the later stages of fruit development. Higher expression of miR156 in later fruit developmental may positively regulate anthocyanin biosynthesis by reducing SPL transcription factor. Novel miRNAs showed variation in expression among different tissues. These novel miRNAs targeted different transcription factors and hormone related regulators. Gene ontology and KEGG pathway analyses revealed predominant metabolic processes and catalytic activities, important for fruit development. In addition, KEGG pathway analyses revealed the involvement of miRNAs in ascorbate and linolenic acid, starch and sucrose metabolism; RNA transport; plant hormone signaling pathways; and circadian clock.ConclusionOur first and preliminary report of miRNAs will provide information on the synthesis of biochemical compounds of pomegranate for future research. The functions of the targets of the novel miRNAs need further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0807-3) contains supplementary material, which is available to authorized users.

Varroa mites, viruses and bacteria incidences in Kenyan domesticated honeybee colonies

ABSTRACTVarroa mite (Varroa destructor Anderson and Trueman) is a major global threat to the western honey bee (Apis mellifera L.). The ectoparasite has been implicated in the spread of honeybee viruses. Beekeeping plays a major role in transmission of the mite. The study aimed at assessing levels of Varroa infestation, bee viruses and bacteria incidences in domesticated honeybee colonies. Samples of adult honey bees, bee brood and Varroa mites were collected from Baringo, Narok, Kwale, Magarini, Voi, Ijara, Busia and Siaya in Kenya. Ten hives in each site were inspected for the presence of Varroa mites on adult bees using the icing sugar technique and forceps in sealed brood cells. The number of mites observed were recorded per site. Ribonucleic acid was extracted from the mites, brood and adult bees and a polymerase chain reaction was performed to detect the black queen cell virus. Pooled RNA samples of brood and adult bees were used in next generation sequencing on a 454 GS FLX platform to detect bee v...

Circular RNA of cattle casein genes are highly expressed in bovine mammary gland

Recent studies have revealed that, in addition to hormones and other protein factors, noncoding RNA molecules play an important regulatory role in milk protein synthesis. Circular RNAs (circRNAs) are universally expressed noncoding RNA species that have been proposed recently to regulate the expression of their parental genes. In the present study, the deep RNA-sequencing technique known as RNA-seq was used to compare expression profiles of circRNAs from 2 pooled RNA samples from cow mammary gland on d 90 and 250 postpartum and to identify the key circRNAs involved in lactation. A total of 4,804 and 4,048 circRNAs were identified in the cow mammary gland on d 90 and 250 postpartum, respectively, of which only 2,231 circRNAs were co-expressed at both lactation stages, suggesting high stage specificity in the circRNAs. The enrichment of some Gene Ontology terms for the circRNA parental genes differed between lactation stages. Among the top 10 enriched Gene Ontology terms, vesicle, endoplasmic reticulum, and mitochondrial lumen were more common on lactation d 90. All 4 casein-coding genes (CSN1S1, CSN1S2, CSN2, and CSN3) produced circRNAs in the cattle mammary gland. In total, 80 circRNAs were identified from these 4 genes; circRNAs from CSN1S1 had very high abundance, and 3 of them accounted for 36% of all the circRNAs expressed in the mammary gland on lactation d 90. Three circRNAs from CSN1S1, 1 circRNA from CSN1S2, and 1 circRNA from CSN2 were all more highly expressed on lactation d 90 than on lactation d 250, as confirmed by quantitative PCR. These circRNAs had several target sites for the microRNA miR-2284 family and were predicted to target CSN1S1 and CSN2 mRNA, suggesting their potential involvement in regulating expression of the casein genes.

Genome-wide expression profiles of Pyropia haitanensis in response to osmotic stress by using deep sequencing technology.

BackgroundPyropia haitanensis is an economically important marine crop grown in harsh intertidal habitats of southern China; it is also an excellent model system for studying mechanisms of stress tolerance. To understand the molecular mechanisms underlying osmotic tolerance and adaptation to intertidal environments, a comprehensive analysis of genome-wide gene expression profiles in response to dehydration and rehydration in Py. haitanensis was undertaken using digital gene expression profile (DGE) approaches combined with de novo transcriptome sequencing.ResultsRNA-sequencing of the pooled RNA samples from different developmental phases and stress treatments was performed, which generated a total of 47.7 million clean reads. These reads were de novo assembled into 28,536 unigenes (≥200 bp), of which 18,217 unigenes (63.83 %) were annotated in at least one reference database. DGE analysis was performed on four treatments (two biological replicates per treatment), which included moderate dehydration, severe dehydration, rehydration, and normal conditions. The number of raw reads per sample ranged from 12.47 to 15.79 million, with an average of 14.69 million reads per sample. After quality filtering, the number of clean reads per sample ranged from 11.83 to 15.04 million. All distinct sequencing reads were annotated using the transcriptome of Py. haitanensis as reference. A total of 1,681 unigenes showed significant differential expression between moderate dehydration and normal conditions, in which 977 genes were upregulated, and 704 genes were downregulated. Between severe dehydration and normal conditions, 1,993 unigenes showed significantly altered expression, which included both upregulated (1,219) and downregulated genes (774). In addition, 1,086 differentially expressed genes were detected between rehydration and normal conditions, of which 720 genes were upregulated and 366 unigenes were downregulated. Most gene expression patterns in response to dehydration differed from that of rehydration, except for the synthesis of unsaturated fatty acids, several transcription factor families, and molecular chaperones that have been collectively implicated in the processes of dehydration and rehydration in Py. haitanensis.ConclusionsTaken together, these data provide a global high-resolution analysis of gene expression changes during osmotic stress that could potentially serve as a key resource for understanding the biology of osmotic acclimation in intertidal red seaweed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2226-5) contains supplementary material, which is available to authorized users.

Oral administration of Lactobacillus plantarum 299v modulates gene expression in the ileum of pigs: prediction of crosstalk between intestinal immune cells and sub-mucosal adipocytes.

To study host–probiotic interactions in parts of the intestine only accessible in humans by surgery (jejunum, ileum and colon), pigs were used as model for humans. Groups of eight 6-week-old pigs were repeatedly orally administered with 5 × 1012 CFU Lactobacillus plantarum 299v (L. plantarum 299v) or PBS, starting with a single dose followed by three consecutive daily dosings 10 days later. Gene expression was assessed with pooled RNA samples isolated from jejunum, ileum and colon scrapings of the eight pigs per group using Affymetrix porcine microarrays. Comparison of gene expression profiles recorded from L. plantarum 299v-treated pigs with PBS-treated pigs indicated that L. plantarum 299v affected metabolic and immunological processes, particularly in the ileum. A higher expression level of several B cell-specific transcription factors/regulators was observed, suggesting that an influx of B cells from the periphery to the ileum and/or the proliferation of progenitor B cells to IgA-committed plasma cells in the Peyer’s patches of the ileum was stimulated. Genes coding for enzymes that metabolize leukotriene B4, 1,25-dihydroxyvitamin D3 and steroids were regulated in the ileum. Bioinformatics analysis predicted that these metabolites may play a role in the crosstalk between intestinal immune cells and sub-mucosal adipocytes. Together with regulation of genes that repress NFKB- and PPARG-mediated transcription, this crosstalk may contribute to tempering of inflammatory reactions. Furthermore, the enzyme adenosine deaminase, responsible for the breakdown of the anti-inflammatory mediator adenosine, was strongly down-regulated in response to L. plantarum 299v. This suggested that L. plantarum 299v-regulated production of adenosine by immune cells like regulatory T cells may also be a mechanism that tempers inflammation in the ileum, and perhaps also in other parts of the pig’s body.Electronic supplementary materialThe online version of this article (doi:10.1007/s12263-015-0461-7) contains supplementary material, which is available to authorized users.

Abstract 5226: Liver and blood miRNA alterations as putative biomarkers of hepatotoxic response to short-term furan exposure in mice

Abstract Epigenetic miRNA-based changes measured in accessible matrices may serve as useful biomarkers of environmental exposures and human health effects. We investigated miRNA profiles following short-term exposure to a known cytotoxic hepatocarcinogen, furan. We measured global liver and blood miRNA changes in female B3C6F1 mice exposed to furan for 3 weeks p.o. at daily doses of 0, 1, 2, 4, and 8 mg/kg. Small RNA was extracted from frozen samples using Exiqon miRCURY RNA Isolation kit and quantified by Qubit. RNA quality was checked by Nanodrop and Bioanalyzer, and liver miRNA were measured by miRNA-seq on the Illumina HiScanSQ platform. Blood miRNA were measured from pooled RNA samples by Affymetrix GeneChip v3 microarrays. In the liver, the 1, 2, 4, and 8 mg/kg doses resulted in 0, 10, 17 and 14 differentially altered miRNAs, respectively (&amp;gt;1.5-fold; B-H corrected p-value&amp;lt;0.05). The majority of miRNA changes in the low-dose exposure group (2mg/kg) were not seen with other treatments (7 of 10 miRNA). Conversely, most altered miRNA observed with high-dose (4 or 8 mg/kg) exposures were shared (10 miRNA), indicating different miRNA-mediated pathways for non-carcinogenic and carcinogenic furan exposures. Using Ingenuity Pathway Analysis (IPA), liver miRNAs altered by the carcinogenic doses of furan (4 and 8 mg/kg) significantly enriched (B-H corrected p-value&amp;lt;0.05) functions related to cellular development, growth and proliferation, movement, cell cycle, death and survival; other categories identified by IPA were related to hepatotoxicity, liver inflammation, and cancer. Using predicted miRNA-mRNA interactions, 68 mRNAs were expressed in a contrasting pattern to miRNA expression (upregulated miRNA/downregulated mRNAs and vice versa). Associated mRNAs showed enrichment of pathways similar to miRNAs. Measurements in whole blood samples indicated that 6 out of the 21 miRNAs altered by the carcinogenic doses of furan were also altered in the blood (4 in the same direction). Blood miRNAs included mmu-miR-34a, -146b, -183, -5099 (upregulated), and -10a, -99b (downregulated). Results demonstrate distinctive miRNA profiles in rodent liver for carcinogenic doses of furan with corresponding changes for a subset of liver miRNAs in blood. Our findings support ongoing efforts to identify novel miRNA biomarkers in accessible matrices related to environmental health effects. This abstract does not necessarily reflect the policy of the US EPA. Citation Format: Brian Norris Chorley, Gail Nelson, Gleta Carswell, Holly Mortensen, James Crooks, William Ward, Charles Wood, Anna F. Jackson, Carole Yauk, Les Recio, Susan Hester. Liver and blood miRNA alterations as putative biomarkers of hepatotoxic response to short-term furan exposure in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5226. doi:10.1158/1538-7445.AM2014-5226

Serum miRNA profiling identifies miR-150/30a as potential biomarker for workers with damaged nerve fibers from carbon disulfide.

As crucial small regulatory molecules, serum microRNAs (miRNAs) have been widely identified as potential noninvasive biomarkers. To survey and identify serum miRNAs associated with workers who had experienced injury to their nerve system from carbon disulfide (CS2), we profiled abnormally expressed miRNAs using the microarray technique and further performed qRT-PCR validation in case and control samples (n=20). Microarray profiling in pooled RNA samples showed that many miRNAs in workers exposed to CS2 were aberrantly expressed. Based on control samples exposed to CS2, a great amount of abnormal miRNAs, including some miRNA gene clusters and families, were obtained from microarray datasets. Most of deregulated miRNAs were up-regulated, and almost all miRNAs showed consistent expression patterns between workers with different numbers of damaged nerve fibers. Functional enrichment analysis suggested that these abnormal miRNAs showed versatile roles by contributing to multiple biological processes. Some aberrantly expressed miRNAs were characterized as miRNA gene clusters or families, and they always showed consistent expression patterns. miR-150 and miR-30a were selected to be further validated by qRT-PCR as up-regulated species, and they could discern case samples from control samples. miR-150 and miR-30a may be potential noninvasive biomarkers for a damaged nervous system.

De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-alzheimer’s properties

BackgroundThe major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid β protein (a pathological marker of Alzheimer’s disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown.ResultsIn this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis.ConclusionA combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria, and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-676) contains supplementary material, which is available to authorized users.

Deep RNA Sequencing Reveals that MicroRNAs Play a Key Role in Lactation in Rats

Understanding the regulatory contribution of maternal physiology to difficulties with lactation is beneficial to both mother and infant. MicroRNAs (miRNAs), a type of noncoding RNA, may be involved in the regulation of mammary gland development and function. In the present study, a deep RNA sequencing (RNA-seq) technique was used to compare the expression profile of miRNAs and mRNAs of 2 pooled RNA samples from day 1 and day 7 postpartum (n = 1/d) rat (Rattus norvegicus) mammary glands to identify key miRNAs and their target genes that may control the rate-limiting steps of lactation. A total of 395 and 400 known miRNAs were identified in days 1 and 7 postpartum rat mammary samples, respectively. Compared with day 1 postpartum, 27 miRNAs were differentially expressed at day 7 postpartum. The expression differences between lactation periods were further analyzed by real-time quantitative polymerase chain reaction (qPCR) (n = 5). The ΔΔCt values of rno-miR-30, rno-miR-1, rno-miR-145-3p, rno-miR-142, rno-miR-7a-5p, rno-miR-3571, rno-miR-224-5p, rno-miR-362-5p, rno-miR-342-3p, rno-miR-322-3p, rno-miR-18a-5p, and rno-miR-202-5p between the 2 libraries varied from 0.64 to 9.44; the ΔΔCt values of rno-miR-133, rno-miR-190a-5p, rno-miR-27a-5p, rno-miR-451-5p, rno-miR-3120, rno-miR-23a-5p, rno-miR-20a-3p, rno-miR-92a-1-5p, and rno-miR-134-5p between the 2 libraries varied from −1.02 to −4.37 (P < 0.05). The intersection of the expressed mRNA genes from RNA-seq and putative target genes of differentially expressed miRNAs, termed mammary gland target genes (MTGs), was analyzed. The results indicated that 1259 MTGs overlapped between the 2 gene sets. The expression of 14 randomly selected genes of the MTGs was further confirmed by real-time qPCR (R2 = 0.86, P < 0.01). The downregulated MTGs were enriched for the pathways involved in lipid biosynthesis. This gene cluster included 24 lipid metabolic process–related genes, which were putative targets of 10 differentially expressed miRNAs. These results will be helpful in discovering the biologic underpinnings of poor lactation performance in women attempting to breastfeed.

Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland.

MicroRNA repertoire for functional genome research in tilapia identified by deep sequencing.

The Nile tilapia (Oreochromis niloticus; Cichlidae) is an economically important species in aquaculture and occupies a prominent position in the aquaculture industry. MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression involved in diverse biological and metabolic processes. To increase the repertoire of miRNAs characterized in tilapia, we used the Illumina/Solexa sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the different developmental stages of tilapia. Bioinformatic analyses suggest that 197 conserved and 27 novel miRNAs are expressed in tilapia. Sequence alignments indicate that all tested miRNAs and miRNAs* are highly conserved across many species. In addition, we characterized the tissue expression patterns of five miRNAs using real-time quantitative PCR. We found that miR-1/206, miR-7/9, and miR-122 is abundantly expressed in muscle, brain, and liver, respectively, implying a potential role in the regulation of tissue differentiation or the maintenance of tissue identity. Overall, our results expand the number of tilapia miRNAs, and the discovery of miRNAs in tilapia genome contributes to a better understanding the role of miRNAs in regulating diverse biological processes.

Genome-Wide Identification of Molecular Pathways and Biomarkers in Response to Arsenic Exposure in Zebrafish Liver

Inorganic arsenic is a worldwide metalloid pollutant in environment. Although extensive studies on arsenic-induced toxicity have been conducted using in vivo and in vitro models, the exact molecular mechanism of arsenate toxicity remains elusive. Here, the RNA-SAGE (serial analysis of gene expression) sequencing technology was used to analyse hepatic response to arsenic exposure at the transcriptome level. Based on more than 12 million SAGE tags mapped to zebrafish genes, 1,444 differentially expressed genes (750 up-regulated and 694 down-regulated) were identified from a relatively abundant transcripts (>10 TPM [transcripts per million]) based on minimal two-fold change. By gene ontology analyses, these differentially expressed genes were significantly enriched in several major biological processes including oxidation reduction, translation, iron ion transport, cell redox, homeostasis, etc. Accordingly, the main pathways disturbed include metabolic pathways, proteasome, oxidative phosphorylation, cancer, etc. Ingenity Pathway Analysis further revealed a network with four important upstream factors or hub genes, including Jun, Kras, APoE and Nr2f2. The network indicated apparent molecular events involved in oxidative stress, carcinogenesis, and metabolism. In order to identify potential biomarker genes for arsenic exposure, 27 out of 29 up-regulated transcripts were validated by RT-qPCR analysis in pooled RNA samples. Among these, 14 transcripts were further confirmed for up-regulation by a lower dosage of arsenic in majority of individual zebrafish. Finally, at least four of these genes, frh3 (ferrintin H3), mgst1 (microsomal glutathione S-transferase-like), cmbl (carboxymethylenebutenolidase homolog) and slc40a1 (solute carrier family 40 [iron-regulated transporter], member 1) could be confirmed in individual medaka fish similarly treated by arsenic; thus, these four genes might be robust arsenic biomarkers across species. Thus, our work represents the first comprehensive investigation of molecular mechanism of asenic toxicity and genome-wide search for potential biomarkers for arsenic exposure.

Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling

The whole zebrafish embryo model (ZFE) has proven its applicability in developmental toxicity testing. Since functional hepatocytes are already present from 36 h post fertilization onwards, whole ZFE have been proposed as an attractive alternative to mammalian in vivo models in hepatotoxicity testing. The goal of the present study is to further underpin the applicability of whole ZFE for hepatotoxicity testing by combining histopathology and next-generation sequencing-based gene expression profiling. To this aim, whole ZFE and adult zebrafish were exposed to a set of hepatotoxic reference compounds. Histopathology revealed compound and life-stage-specific effects indicative of toxic injury in livers of whole ZFE and adult zebrafish. Next-generation sequencing (NGS) was used to compare transcript profiles in pooled individual RNA samples of whole ZFE and livers of adult zebrafish. This revealed that hepatotoxicity-associated expression can be detected beyond the overall transcription noise in the whole embryo. In situ hybridization verified liver specificity of selected highly expressed markers in whole ZFE. Finally, cyclosporine A (CsA) was used as an illustrative case to support applicability of ZFE in hepatotoxicity testing by comparing CsA-induced gene expression between ZFE, in vivo mouse liver and HepaRG cells on the levels of single genes, pathways and transcription factors. While there was no clear overlap on single gene level between the whole ZFE and in vivo mouse liver, strong similarities were observed between whole ZFE and in vivo mouse liver in regulated pathways related to hepatotoxicity, as well as in relevant overrepresented transcription factors. In conclusion, both the use of NGS of pooled RNA extracts analysis combined with histopathology and traditional microarray in single case showed the potential to detect liver-related genes and processes within the transcriptome of a whole zebrafish embryo. This supports the applicability of the whole ZFE model for compound-induced hepatotoxicity screening.

Isolation and Functional Analysis of GarCIPK8 Gene from Gossypium aridum

CIPK (calcineurin B-like calcium sensor interacting protein kinase) is a typical group of serine/threonine protein kinase targeting calcium sensor calcineurin B-like protein-specific. In a previous study, we obtained the transcriptome of Gossypium aridum with a pooled RNA sample from root and leaf of G. aridum under salt stress. From the transcriptome data, we founded that a CIPK related gene had high level of expression abundance. Real-time fluorescence quantitative PCR analysis showed its relative expression level was up-regulated in root induced by salt stress, indicating this CIPK related gene might be involved in response to salt stress during cotton seedling stage. By in silico cloning and RT-PCR technology, a full-length cDNA encoding a CIPKs homologue (GarCIPK8) was isolated from G. aridum. The deduced GarCIPK8 protein had 449 amino acids with a predicted molecular weight of 51.12 kD and a predicted isoelectric point of 8.13. The kinase N-terminal catalytic domain of GarCIPK8 exhibited a typica1 serine/threonine protein kinase domain. The conserved 24 amino acid motif existed in C-terminal non-kinase regions of GarCIPK8 protein. Alignment of amino acid sequence showed that GarCIPK8 was 73.95% identical to Oryza sativa OsCIPK8 protein. In order to characterize its putative function, the GarCIPK8 gene driven by CaMV35S promoter and stress-specific promoter rd29A respectively were transformed into tobacco by Agrobacterium-mediated transgenic technology. PCR and RT-PCR detection confirmed that GarCIPK8 gene was integrated into tobacco genome and expressed normally. The growth of transgenic plants of T1 generation was observed under salinity and drought stresses, showing that transgenic plants enhance tolerance to both high salt and osmotic stresses.