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Technical tips, new applications, and product highlights from Epicentre Biotechnologies.
Umland et al. investigated essential genes for growth in Acinetobacter baumannii by generating mutants using the EZ-Tn5™ KAN-2 Transposome. In the past, other researchers have found EZ-Tn5 transposomes extremely useful in characterizing the genetics of this bacterium.
A subset of 34 mutants with unique gene disruptions that demonstrated little to no growth on ascites underwent evaluation in a rat subcutaneous abscess model, and these results established that 18 (53%) of these genes could be ........ Read more »
Umland TC et al. (2012) In Vivo-Validated Essential Genes Identified in Acinetobacter baumannii by Using Human Ascites Overlap Poorly with Essential Genes Detected on Laboratory Media. mBio, 3(4). PMID: 22911967
(See Part 1)
A large archive of FFPE tumor samples exists globally; many are readily cross-referenced to clinical records. A wealth of information is thus potentially available to search for biomarkers among expressed genes using whole-transcriptome profiling. However, RNA from FFPE samples is generally severely degraded and, in the absence of enrichment methods, rRNA reads account for a large portion of the deep sequencing information.
Epicentre's Ribo-Zero™ kits provide a reliable method........ Read more »
Morlan JD et al. (2012) Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue. PloS one, 7(8). PMID: 22900061
Rapid Amplification of cDNA Ends (RACE), the method of choice to study RNA transcription start sites, can be readily accomplished using enzymatic processing of RNA for downstream PCR. Kim et al. report a genome-wide survey of transcription start sites (TSS) in the pathogenic bacteria Escherichia coli and Klebsiella pneumoniae.
The method used two unique Epicentre enzymes, RNA 5'-Polyphosphatase and Terminator Exonuclease, as part of a procedure to isolate and process mRNA from bacterial tota........ Read more »
Kim D et al. (2012) Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling. PLoS Genetics, 8(8). PMID: 22912590
The EZ-Tn5™ Transposome System is now finding favor in studies of more unusual environmental microbial species. The bacterial phylum Planctomycetes is found in various environmental habitats, as well as in the gastrointestinal tract of many mammals and fish. Members of this phylum possess characteristics that make them, at first glance, unsuitable for in vivo transposition experiments to develop a model genetic system. Although genome sequencing data are available for several Planctomycetes ........ Read more »
Schreier HJ et al. (2012) Transposon Mutagenesis of Planctomyces limnophilus and Analysis of a pckA Mutant. Appl Environ Microbiol. PMID: 22798371
Ritlop et al. describe the use of a custom-engineered EZ-Tn5™ transposon and a powerful genetic screen to investigate the autocatalytic splicing of group II introns from pre-mRNA. The self-splicing 3.5-kb L1.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was the subject of the study.
The custom transposon contains a pepN transcriptional promoter followed by the P23 L. lactis constitutive promoter. In vitro transposition into a cloned gene created a library of clones ........ Read more »
Ritlop, C. et al. (2012) Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen. PLoS ONE, 7(8). DOI: 10.1371/journal.pone.0041589
Stadler et al. investigated the molecular effects of miRNA regulation on certain well-characterized C. elegans genes using high-throughput ribosome profiling, a novel method to study translation of mRNAs and translation control by miRNAs. The method uses Epicentre's Circligase™ enzyme to generate sequencing template (see Figures 1 and 5 in Epicentre's poster: 1.8 MB PDF).
Briefly, ribosomes were isolated and concentrated by sucrose-gradient centrifugation, and treated with a ribonuclease to ........ Read more »
Stadler M et al. (2012) Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets. Genome Res. PMID: 22855835
Many FFPE tissue blocks containing samples related to known disease states are archived in a multitude of hospitals worldwide. The RNA available from these blocks is not only limited in quantity but also generally severely degraded. In this report, Sinicropi et. al., discuss methods for RNA-Seq using RNA isolated from archival FFPE samples with low input levels of RNA, and compare the results to data previously generated by RT-PCR analysis.
The authors used the MasterPure™ RNA Pur........ Read more »
Sinicropi D et al. (2012) Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue. PloS one, 7(7). PMID: 22808097
Multiple sclerosis (MS) is a devastating illness with no known cause or cure. Recently, Laska et al. revealed that onset of the illness appears to increase expression of a specific retrovirus, HERV-Fc1, that belongs to the HERV-H/F family.
The study included investigating expression of certain components of HERV-Fc1 by qRT-PCR. During the studies, standards for various expressed transcripts of the virus were generated using the Durascribe® T7 Transcription Kit. The Durascribe Kit synthesizes R........ Read more »
Laska MJ et al. (2012) Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active multiple sclerosis. Journal of virology, 86(7), 3713-22. PMID: 22278236
EZ-Tn5™ Transposomes are popular for generation of insertion and knockout mutants in microorganisms, 12 years after their introduction. A recent report by Veeranagouda et al. describes the generation of mutants in the anaerobe Bacteroides fragilis. The study is notable since anaerobes tend to be quite difficult to mutagenize through transposition, due to electroporation in the presence of oxygen that can poison the organism.
The experiments performed on B. fragilis used a custom transposome........ Read more »
Veeranagouda, Y et al. (2012) Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome. FEMS Microbiology Letters. PMID: 22639975
Genomic analysis has greatly increased our understanding of bacterial-insect interactions. Yet there remains a need for effective methods to remove insect rRNA to enable comprehensive, transcriptome-based studies. In a recent publication, Kumar et al. investigated the performance of the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) with insect samples. Eggs derived from a horizontal gene transfer of the Wolbachia bacterial genome into Drosophila ananassae were chosen as a model system. The ........ Read more »
Kumar, N. et al. (2012) Efficient subtraction of insect rRNA prior to transcriptome analysis of Wolbachia-Drosophila lateral gene transfer. BMC Res Notes, 230. DOI: 10.1186/1756-0500-5-230
The EZ-Tn5™ R6Kγori/KAN-2 Transposome has been used for elucidation of bacterial gene structure and function for the last 12 years. In a recent publication by Khatiwara et al., it was used to mutagenize a strain of Salmonella enterica serotype Typhimurium to link specific gene expression information to the gene of interest. The mutagenesis work also demonstrates a new method of transposome insertion sequencing using Illumina technology instead of older Sanger sequencing techniques.
The resea........ Read more »
Khatiwara, A. et al. (2012) Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium. Applied and Environmental Microbiology, 78(9), 3098-3107. DOI: 10.1128/AEM.06865-11
Microbial geneticists continue to use the EZ-Tn5 in vivo transposomics tools for establishing genetic systems in novel bacteria. Jogler et al. report on the Planctomycetes phylum of bacteria which is a major component of the global nitrogen and carbon cycles. These bacteria perform reactions such as the anaerobic oxidation of ammonium ions. Many Planctomycetes strains are difficult to cultivate, so the researchers developed a model system using the P. limnophilus strain due to its cultivabilit........ Read more »
Jogler, C. et al. (2011) Characterization of Planctomyces limnophilus and Development of Genetic Tools for Its Manipulation Establish It as a Model Species for the Phylum Planctomycetes. Applied and Environmental Microbiology, 77(16), 5826-5829. DOI: 10.1128/AEM.05132-11
The Tasmanian devil was recently listed as an endangered species, primarily due to the emergence of a fatal, transmissible cancer known as devil facial tumour disease (DFTD). The disease was recently reported in Northern Tasmania, and has resulted in depletion of populations that could ultimately make the animals extinct within 25 to 35 years. Recently, Deakin et al. conducted a genetic survey of a BAC library created with the CopyControl™ BAC Library Construction Kit (EcoR I) from genomic DNA........ Read more »
Deakin, J. et al. (2012) Genomic Restructuring in the Tasmanian Devil Facial Tumour: Chromosome Painting and Gene Mapping Provide Clues to Evolution of a Transmissible Tumour. PLoS Genetics, 8(2). DOI: 10.1371/journal.pgen.1002483
The role of DNA methylation in gene regulation and epigenetics is of ever-increasing interest. Until now, investigating DNA methylation has been crippled either by: i) the necessity for microgram quantities of input DNA for whole-genome bisulfite sequencing (WGBS); or ii) incomplete interrogation of the methylome in the case of reduced-representation bisulfite sequencing (RRBS).
In a recent publication, Adey and Shendure report a transposon-based in vitro library construction method to de........ Read more »
Adey, A., & Shendure, J. (2012) Ultra-low-input, tagmentation-based whole genome bisulfite sequencing. Genome Research. DOI: 10.1101/gr.136242.111
Although much of RNA-Seq research has focused on the human transcriptome, there is considerable interest in applications of RNA-Seq to environmental metatranscriptomics samples. Giannoukos et al. at the Broad Institute evaluated several methods for removal of ribosomal RNA (rRNA) from total RNA preparations of complex microbial communities in order to maximize RNA-Seq reads. They also examined bacterial populations from clinical human stool samples.
The researchers used total RNA from well-........ Read more »
Giannoukos, G. et al. (2012) Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes. Genome Biology, 13(3). DOI: 10.1186/gb-2012-13-3-r23
A recent study by Koerner et al. cites the use of Epicentre's most popular products: the Ribo-Zero™ Kit (Human/Mouse/Rat) to deplete rRNA, and the ScriptSeq™ Kit to prepare RNA-Seq libraries. The authors explored the presence of CpG islands at the 5' end of the Airn noncoding RNA (ncRNA). This region contains some unusual features, such as a GC-rich region just downstream from the promoter, and tandem direct repeats (TDR) in its second half.
The authors used homologous recombination to gen........ Read more »
Koerner, M. et al. (2012) A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter. PLoS Genetics, 8(3). DOI: 10.1371/journal.pgen.1002540
Transposon mutagenesis is still considered to be an excellent tool for probing the genomics of bacterial species to create gene-silencing mutations. This approach can help elucidate the genetic markers responsible for various bioactivities.
A report by Garavaglia et al. explores the pathway of biosynthetic genes responsible for biofilm formation in the bacterium E. coli MG1655. Using the EZ-Tn5™ <R6Kγori/KAN-2> Transposome, insertional mutants were characterized that allowed the rese........ Read more »
Garavaglia, M. et al. (2012) The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli. PLoS ONE, 7(2). DOI: 10.1371/journal.pone.0031252
Göransson et al. report a new method of finding pathogens as part of a biosecurity study that is able to detect pathogens down to the single-molecule/organism level. The method combines a padlock probe approach, using Ampligase® Thermostable DNA Ligase, with rolling-circle amplification (RCA).
The authors used an environmental air sampling unit to trap particulate material on a membrane, followed by a rapid extraction of the DNA using magnetic beads. After clean-up, the DNA-containing sol........ Read more »
Göransson, J. et al. (2012) Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification. PLoS ONE, 7(2). DOI: 10.1371/journal.pone.0031068
RNA-Seq techniques are well integrated into the study of cancer genetics and are useful in locating splice variants in genes that may be indicative of a malignant cell. In a recent study, Salzman et al. observed, using RNA-Seq analysis, that a large amount of spliced transcripts isolated from cancer cells are actually circular in nature. The results suggest that other splicing modes are possible and may be useful in differentiating cancerous from normal tissue types. So-called "scrambled exons",........ Read more »
Salzman, J. et al. (2012) Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types. PLoS ONE, 7(2). DOI: 10.1371/journal.pone.0030733
RNAse R is a unique Epicentre enzyme that is finding greater use in studying single-stranded RNAs, including circular RNAs that contain linear single protruding strands ("lariats"). These molecules have important biological functions, including roles in viral life cycles and tRNA maturation .However, discovery of circular RNAs has so far been mostly serendipitous, and methods to study these molecules are needed.
Danan et al. developed a directed method to pinpoint RNA-Seq reads that have a per........ Read more »
Danan, M. et al. (2011) Transcriptome-wide discovery of circular RNAs in Archaea. Nucleic Acids Research. DOI: 10.1093/nar/gkr1009
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