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Items: 10

1.
BMC Res Notes. 2015 Dec 8;8:754. doi: 10.1186/s13104-015-1726-3.

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.

Author information

1
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. heraadaas@gmail.com.
2
Unit of Biochemistry, Faculty of Medicine, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. heraadaas@gmail.com.
3
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. sivachandranparimannan@gmail.com.
4
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. cvsureshgupta@gmail.com.
5
Malaysian Genomics Resource Centre, 27-9, Level 9 Boulevard Signature Offices, 59200, Mid Valley City, Malaysia. Joanne.mason@ouh.nhs.uk.
6
Oxford Biomedical Research Centre, Old Road Headington Oxford, Oxfordshire, OX3 7LE, UK. Joanne.mason@ouh.nhs.uk.
7
Malaysian Genomics Resource Centre, 27-9, Level 9 Boulevard Signature Offices, 59200, Mid Valley City, Malaysia. laurence@mgrc.com.my.
8
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. ravichandran@aimst.edu.my.
9
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. su_yin@aimst.edu.my.

Abstract

BACKGROUND:

Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform.

RESULTS:

TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique.

CONCLUSIONS:

Amresco's Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.

PMID:
26645211
PMCID:
PMC4673735
DOI:
10.1186/s13104-015-1726-3
[Indexed for MEDLINE]
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2.
Cancer Res. 2016 Jan 15;76(2):319-28. doi: 10.1158/0008-5472.CAN-15-0751. Epub 2015 Nov 10.

Gender-Specific Molecular and Clinical Features Underlie Malignant Pleural Mesothelioma.

Author information

1
The Thoracic Surgery Oncology laboratory and the International Mesothelioma Program, Division of Thoracic Surgery and the Lung Center, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts. aderienzo@partners.org.
2
The Thoracic Surgery Oncology laboratory and the International Mesothelioma Program, Division of Thoracic Surgery and the Lung Center, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts.
3
Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
4
Center for Cancer Computational Biology, Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, and Harvard School of Public Health, Boston, Massachusetts.
5
Departments of Pathology, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts.
6
Malaysian Genomics Resource Centre, Kuala Lumpur, Malaysia.
7
Debakey Department of Surgery, Baylor College of Medicine, Houston, Texas.

Abstract

Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs more frequently in men, but is associated with longer survival in women. Insight into the survival advantage of female patients may advance the molecular understanding of MPM and identify therapeutic interventions that will improve the prognosis for all MPM patients. In this study, we performed whole-genome sequencing of tumor specimens from 10 MPM patients and matched control samples to identify potential driver mutations underlying MPM. We identified molecular differences associated with gender and histology. Specifically, single-nucleotide variants of BAP1 were observed in 21% of cases, with lower mutation rates observed in sarcomatoid MPM (P < 0.001). Chromosome 22q loss was more frequently associated with the epithelioid than that nonepitheliod histology (P = 0.037), whereas CDKN2A deletions occurred more frequently in nonepithelioid subtypes among men (P = 0.021) and were correlated with shorter overall survival for the entire cohort (P = 0.002) and for men (P = 0.012). Furthermore, women were more likely to harbor TP53 mutations (P = 0.004). Novel mutations were found in genes associated with the integrin-linked kinase pathway, including MYH9 and RHOA. Moreover, expression levels of BAP1, MYH9, and RHOA were significantly higher in nonepithelioid tumors, and were associated with significant reduction in survival of the entire cohort and across gender subgroups. Collectively, our findings indicate that diverse mechanisms highly related to gender and histology appear to drive MPM.

PMID:
26554828
PMCID:
PMC4715909
DOI:
10.1158/0008-5472.CAN-15-0751
[Indexed for MEDLINE]
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3.
Genome Biol Evol. 2015 Oct 6;7(10):2885-95. doi: 10.1093/gbe/evv186.

Whole Genome Sequencing of the Asian Arowana (Scleropages formosus) Provides Insights into the Evolution of Ray-Finned Fishes.

Author information

1
School of Science, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia Monash University Malaysia Genomics Facility, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia.
2
School of Science, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia Monash University Malaysia Genomics Facility, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia Malaysian Genomics Resource Centre Berhad, Boulevard Signature Office, Kuala Lumpur, Malaysia.
3
Museum and Art Gallery of the Northern Territory, Darwin, NT, Australia.
4
School of Science, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia Monash University Malaysia Genomics Facility, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia gan.han.ming@monash.edu.

Abstract

The Asian arowana (Scleropages formosus) is of commercial importance, conservation concern, and is a representative of one of the oldest lineages of ray-finned fish, the Osteoglossomorpha. To add to genomic knowledge of this species and the evolution of teleosts, the genome of a Malaysian specimen of arowana was sequenced. A draft genome is presented consisting of 42,110 scaffolds with a total size of 708 Mb (2.85% gaps) representing 93.95% of core eukaryotic genes. Using a k-mer-based method, a genome size of 900 Mb was also estimated. We present an update on the phylogenomics of fishes based on a total of 27 species (23 fish species and 4 tetrapods) using 177 orthologous proteins (71,360 amino acid sites), which supports established relationships except that arowana is placed as the sister lineage to all teleost clades (Bayesian posterior probability 1.00, bootstrap replicate 93%), that evolved after the teleost genome duplication event rather than the eels (Elopomorpha). Evolutionary rates are highly heterogeneous across the tree with fishes represented by both slowly and rapidly evolving lineages. A total of 94 putative pigment genes were identified, providing the impetus for development of molecular markers associated with the spectacular colored phenotypes found within this species.

KEYWORDS:

evolutionary rate; fish; genome; phylogenomics; pigmentation genes

PMID:
26446539
PMCID:
PMC4684697
DOI:
10.1093/gbe/evv186
[Indexed for MEDLINE]
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4.
Microbiologyopen. 2012 Dec;1(4):490-501. doi: 10.1002/mbo3.49. Epub 2012 Nov 28.

Extragenic suppressor mutations that restore twitching motility to fimL mutants of Pseudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels.

Author information

1
The ithree institute, University of Technology Sydney, Sydney, New South Wales, 2007, Australia.

Abstract

Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.

PMID:
23233287
PMCID:
PMC3535393
DOI:
10.1002/mbo3.49
[Indexed for MEDLINE]
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5.
BMC Syst Biol. 2012 Jul 23;6:90. doi: 10.1186/1752-0509-6-90.

Multiple independent analyses reveal only transcription factors as an enriched functional class associated with microRNAs.

Author information

1
Center for Non-coding RNA in Technology and Health, Division of Genetics and Bioinformatics, IBHV, University of Copenhagen, Copenhagen, Denmark.

Abstract

BACKGROUND:

Transcription factors (TFs) have long been known to be principally activators of transcription in eukaryotes and prokaryotes. The growing awareness of the ubiquity of microRNAs (miRNAs) as suppressive regulators in eukaryotes, suggests the possibility of a mutual, preferential, self-regulatory connectivity between miRNAs and TFs. Here we investigate the connectivity from TFs and miRNAs to other genes and each other using text mining, TF promoter binding site and 6 different miRNA binding site prediction methods.

RESULTS:

In the first approach text mining of PubMed abstracts reveal statistically significant associations between miRNAs and both TFs and signal transduction gene classes. Secondly, prediction of miRNA targets in human and mouse 3'UTRs show enrichment only for TFs but not consistently across prediction methods for signal transduction or other gene classes. Furthermore, a random sample of 986 TarBase entries was scored for experimental evidence by manual inspection of the original papers, and enrichment for TFs was observed to increase with score. Low-scoring TarBase entries, where experimental evidence is anticorrelated miRNA:mRNA expression with predicted miRNA targets, appear not to select for real miRNA targets to any degree. Our manually validated text-mining results also suggests that miRNAs may be activated by more TFs than other classes of genes, as 7% of miRNA:TF co-occurrences in the literature were TFs activating miRNAs. This was confirmed when thirdly, we found enrichment for predicted, conserved TF binding sites in miRNA and TF genes compared to other gene classes.

CONCLUSIONS:

We see enrichment of connections between miRNAs and TFs using several independent methods, suggestive of a network of mutual activating and suppressive regulation. We have also built regulatory networks (containing 2- and 3-loop motifs) for mouse and human using predicted miRNA and TF binding sites and we have developed a web server to search and display these loops, available for the community at http://rth.dk/resources/tfmirloop.

PMID:
22824421
PMCID:
PMC3430561
DOI:
10.1186/1752-0509-6-90
[Indexed for MEDLINE]
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6.
RNA. 2012 Mar;18(3):472-84. doi: 10.1261/rna.027615.111. Epub 2012 Jan 26.

Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs.

Author information

1
Laboratorio di Cardiologia Molecolare, IRCCS-Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy.

Abstract

In order to understand the role of microRNAs (miRNAs) in vascular physiopathology, we took advantage of deep-sequencing techniques to accurately and comprehensively profile the entire miRNA population expressed by endothelial cells exposed to hypoxia. SOLiD sequencing of small RNAs derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% O₂ or normoxia for 24 h yielded more than 22 million reads per library. A customized bioinformatic pipeline identified more than 400 annotated microRNA/microRNA* species with a broad abundance range: miR-21 and miR-126 totaled almost 40% of all miRNAs. A complex repertoire of isomiRs was found, displaying also 5' variations, potentially affecting target recognition. High-stringency bioinformatic analysis identified microRNA candidates, whose predicted pre-miRNAs folded into a stable hairpin. Validation of a subset by qPCR identified 18 high-confidence novel miRNAs as detectable in independent HUVEC cultures and associated to the RISC complex. The expression of two novel miRNAs was significantly down-modulated by hypoxia, while miR-210 was significantly induced. Gene ontology analysis of their predicted targets revealed a significant association to hypoxia-inducible factor signaling, cardiovascular diseases, and cancer. Overexpression of the novel miRNAs in hypoxic endothelial cells affected cell growth and confirmed the biological relevance of their down-modulation. In conclusion, deep-sequencing accurately profiled known, variant, and novel microRNAs expressed by endothelial cells in normoxia and hypoxia.

PMID:
22282338
PMCID:
PMC3285935
DOI:
10.1261/rna.027615.111
[Indexed for MEDLINE]
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7.
Plant J. 2011 Oct;68(1):159-67. doi: 10.1111/j.1365-313X.2011.04676.x. Epub 2011 Aug 5.

Intron splicing suppresses RNA silencing in Arabidopsis.

Author information

1
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld 4072, Australia.

Abstract

Silencing of introduced transgenes constitutes a major bottleneck in the production of transgenic crops. Commonly, these transgenes contain no introns, a feature shared with transposons, which are also prime targets for gene silencing. Given that introns are very common in endogenous genes but are often lacking in transgenes and transposons, we hypothesised that introns may suppress gene silencing. To investigate this, we conducted a genome-wide analysis of small RNA densities in exons from intronless versus intron-containing genes in Arabidopsis thaliana. We found that small RNA libraries are strongly enriched for exon sequences derived from intronless genes. Small RNA densities in exons of intronless genes were comparable to exons of transposable elements. To test these findings in vivo we used a transgenic reporter system to determine whether introns are able to suppress gene silencing in Arabidopsis. Introducing an intron into a transgene reduced silencing by more than fourfold. Compared with intronless transcripts, the spliced transcripts were less effective substrates for RNA-dependent RNA polymerase 6-mediated gene silencing. This intron suppression of transgene silencing requires efficient intron splicing and is dependent on ABH1, the Arabidopsis orthologue of human cap-binding protein 80.

[Indexed for MEDLINE]
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8.
J Exp Bot. 2011 May;62(8):2495-506. doi: 10.1093/jxb/erq437. Epub 2011 Apr 19.

MicroRNAs in the shoot apical meristem of soybean.

Author information

1
ARC Centre of Excellence for Integrative Legume Research, Faculty of Land and Food Resources, The University of Melbourne, Parkville, Victoria, 3010, Australia.

Abstract

Plant microRNAs (miRNAs) play crucial regulatory roles in various developmental processes. In this study, we characterize the miRNA profile of the shoot apical meristem (SAM) of an important legume crop, soybean, by integrating high-throughput sequencing data with miRNA microarray analysis. A total of 8423 non-redundant sRNAs were obtained from two libraries derived from micro-dissected SAM or mature leaf tissue. Sequence analysis allowed the identification of 32 conserved miRNA families as well as 8 putative novel miRNAs. Subsequent miRNA profiling with microarrays verified the expression of the majority of these conserved and novel miRNAs. It is noteworthy that several miRNAs* were expressed at a level similar to or higher than their corresponding mature miRNAs in SAM or mature leaf, suggesting a possible biological function for the star species. In situ hybridization analysis revealed a distinct spatial localization pattern for a conserved miRNA, miR166, and its star speciessuggesting that they serve different roles in regulating leaf development. Furthermore, localization studies showed that a novel soybean miRNA, miR4422a, was nuclear-localized. This study also indicated a novel expression pattern of miR390 in soybean. Our approach identified potential key regulators and provided vital spatial information towards understanding the regulatory circuits in the SAM of soybean during shoot development.

PMID:
21504877
DOI:
10.1093/jxb/erq437
[Indexed for MEDLINE]
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9.
Mol Hum Reprod. 2011 Feb;17(2):92-103. doi: 10.1093/molehr/gaq084. Epub 2010 Oct 8.

Evaluation of polymorphisms in predicted target sites for micro RNAs differentially expressed in endometriosis.

Author information

1
Queensland Institute of Medical Research, Herston, Brisbane, Queensland, Australia. Zhen.Zhao@qimr.edu.au

Abstract

Previous microarray analyses identified 22 microRNAs (miRNAs) differentially expressed in paired ectopic and eutopic endometrium of women with and without endometriosis. To investigate further the role of these miRNAs in women with endometriosis, we conducted an association study aiming to explore the relationship between endometriosis risk and single-nucleotide polymorphisms (SNPs) in miRNA target sites for these differentially expressed miRNAs. A panel of 102 SNPs in the predicted miRNA binding sites were evaluated for an endometriosis association study and an ingenuity pathway analysis was performed. Fourteen rare variants were identified in this study. We found SNP rs14647 in the Wolf-Hirschhorn syndrome candidate gene1 (WHSC1) 3'UTR (untranslated region) was associated with endometriosis-related infertility presenting an odds ratio of 12.2 (95% confidence interval = 2.4-60.7, P = 9.03 × 10(-5)). SNP haplotype AGG in the solute carrier family 22, member 23 (SLC22A23) 3'UTR was associated with endometriosis-related infertility and more severe disease. With the individual genotyping data, ingenuity pathways analysis identified the tumour necrosis factor and cyclin-dependant kinase inhibitor as major factors in the molecular pathways. Significant associations between WHSC1 alleles and endometriosis-related infertility and SLC22A23 haplotypes and the disease severe stage were identified. These findings may help focus future research on subphenotypes of this disease. Replication studies in independent large sample sets to confirm and characterize the involvement of the gene variation in the pathogenesis of endometriosis are needed.

PMID:
20935158
PMCID:
PMC3023296
DOI:
10.1093/molehr/gaq084
[Indexed for MEDLINE]
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10.
BMC Genomics. 2009 Apr 20;10:163. doi: 10.1186/1471-2164-10-163.

A transcriptional sketch of a primary human breast cancer by 454 deep sequencing.

Author information

1
Institute of Biomedical Technologies, National Research Council, Milan, Italy. alessandro.guffanti@genomnia.com

Abstract

BACKGROUND:

The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts.

RESULTS:

We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas.

CONCLUSION:

Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling.

PMID:
19379481
PMCID:
PMC2678161
DOI:
10.1186/1471-2164-10-163
[Indexed for MEDLINE]
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