Tuesday, December 17, 2013

Select the Right Tool for your Molecular Biology Project

Select the right tool for your molecular biology project with the new Molecular Biology Selection Guide. This guide enables you to see all your available options for each step of your workflow. Tools are available for sequencing, microarrays, PCR, cloning, and more. 

Wednesday, November 6, 2013

RNA aptamers:  A new paradigm in cancer diagnosis and treatment

RNA aptamers are small, single-stranded RNA molecules that are functionally analogous to antibodies.  Interestingly, they are emerging as a class of molecules with potential in the diagnosis and treatment of human diseases.

Epicentre will host a Free Webinar on Nov 7/13 that will focus on the use of RNA aptamers for developing cancer diagnostics and treatments:

  • Selecting and using RNA aptamers as therapeutic agents
  • Targeted delivery of therapeutics such as siRNA
  • Development of biosensors/diagnostics for early cancer detection

We hope you can join us for this informative session. 

Wednesday, October 23, 2013

Visit Epicentre at ASHG

Epicentre will be attending ASHG to be held October 23-25, in Boston, MA. Stop by Booth #528 to learn more about our products for Methyl-Seq, ribosome profiling, and more.

In addition, we will be presenting the following posters:

  • 477F: Whole genome methylation analysis using a novel bisulfite sequencing scheme.
  • 622F: Understanding translational regulation using RNA-Seq of Ribosome Protected mRNA fragments.

We hope to see you at the conference! If you're not attending, and would like more information about the products highlighted at the conference, please contact us by e-mail or call 1 (800) 284-8474 within the US.

Wednesday, October 2, 2013

EpiGnome™ Methyl-Seq Kit: Informatics Guide Now Available

Sequencing-based DNA methylation analysis applies the coverage density and flexibility enabled by next-generation sequencing to enhance epigenetic studies. Analysis of the sequencing data is an important step of the procedure and so an Informatics Guide is available to get started. The guide produces an output file that can be viewed in a genome viewer and contains sequence information.

Click here for the Informatics Guide

EpiGnome™ Methyl-Seq Kit: Sample Data Set Now Available

The following test data set may be downloaded for use with the Methyl-Seq BioInformatics Guide. This data set was generated using EpiGnome Methyl-Seq Kit with 50 ng of Coriell gDNA (GM12878) as input into bisulfite conversion. The libraries were sequenced PE 75 bp reads. The data set contains 10,000 reads from Read 1 and Read 2.

Click here for the sample data set

Thursday, September 5, 2013

Complete methylome analysis from only 50 ng

Epicentre's new Methyl-Seq Kit produces whole genome bisulfite sequencing libraries from only 50 ng of genomic DNA. EpiGnome™ follows a unique "post-bisulfite conversion" library construction method which yields highly diverse libraries with uniform coverage.

  • Highly diverse libraries
  • Uniform CpG, CHG, and CHH coverage
  • 1-day procedure
  • Only 50 ng into bisulfite-conversion

Thursday, August 15, 2013

Successful PCR Optimization with FailSafe™

We recently received a praiseworthy testimonial for FailSafe PCR System from one of our customers at the Kennedy Institute of Rheumatology, at the University of Oxford, and thought it was worth sharing. The following submission appears in its original form, and reports of successful amplification using FailSafe™ 2X PCR PreMix D for long amplicons.

At the start of my project I had to overcome the difficulty of successfully amplifying long PCR products. I had previously used a product system specifically stated to be capable of expanding long PCR products; however, this approach was unsuccessful. The number of samples which required genotyping was increasing while I spent time adjusting the parameters of the PCR protocol, and the ability to correctly genotype my samples was critical to the progress of my research.  The decision was made to optimise the components of the PCR reaction mix.

Previous work in my laboratory involved the use of pre-mixed ‘FailSafe’ PCR buffers from Epicentre, containing all of the reagents required for PCR, with the exception of the template DNA and DNA polymerase. Epicentre FailSafe buffer D had previously been successfully used to amplify long PCR products. This buffer was incorporated into the protocol for PCR amplicon of long PCR amplicon and resulted in successful amplification compared to the competitor’s product (A). This result made it possible to identify the genotype of individual samples (B). I also used Epicentre ‘FailSafe’ PCR Buffer J to successfully and reproducibly expand smaller PCR amplification products.

These FailSafe buffers resolved my genotyping issues and have been working perfectly since I began using them. I would recommend them to other researchers who would like to simplify and resolve problems with their PCR procedures.

Tuesday, July 23, 2013

Introducing: ScriptSeq™ Complete Gold Kit (Yeast)

Introducing: ScriptSeq™ Complete Gold Kit (Yeast)
The ScriptSeq™ Complete Gold Kit (Yeast) combines Epicentre's powerful Ribo-Zero™ technology and rapid ScriptSeq v2 RNA-Seq library prep procedure for optimized comprehensive preparation of RNA-Seq libraries in 1 day (Fig. 1). The kit requires 1-5 µg of yeast total RNA and contains Ribo-Zero rRNA removal reagents for removal of 25/28S,18S, 5.8S, and 5S rRNA along with 21S-mt and 15S-mt rRNA from yeast total RNA, along with the ScriptSeq v2 RNA-Seq kit for directional RNA-Seq.

Figure 1. An overview of the ScriptSeq™ Complete Gold Kit (Yeast) process. Cluster-ready ScriptSeq RNA-Seq libraries, virtually rRNA-free, are made in 1 day from as little as 100 ng of intact or partially degraded total RNA.

Tuesday, June 4, 2013

Characterizing biofilm formation with the FailSafe™ PCR System

The Hha gene in E. coli encodes the haemolysin expression modulating protein, a gene product that is responsible for pathogenesis in Enterohemorrhagic E. coli (EHEC) strain O157:H7 - a common pathogenic organism that is found in biofilms in hospital medical environments. While molecular mechanisms promoting adherence of this strain on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In order to more fully understand how the Hha gene regulates biofilm formation, researchers at the USDA generated mutant forms of three different E. coli genes, including Hha, using the FailSafe™ PCR System. A hha deletion mutant was developed using deletion recombination to remove the sequence of the Hha open reading frame from the bacterium. The removal of the Hha sequence also had other consequences: the deletion mutant produced large quantities of biofilm compared to the O157:H7 wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced in the hha mutants, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind dye Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively.
ResearchBlogging.orgSharma VK et al. (2013). Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD. Applied and environmental microbiology, 79 (7), 2384-96 PMID: 23377937

Thursday, April 25, 2013

Introducing: TotalScript™ RNA-Seq Kit

RNA-seq without rRNA removal
The TotalScript Kit prepares high complexity RNA-Seq libraries from only 1-5 ng of intact total RNA, without the need for poly(A) enrichment or rRNA removal. Powered by Nextera™, TotalScript is index-capable, produces directional RNA-seq libraries, and is ideal for high quality RNA (RIN >7) from human, rodent, yeast, C. elegans and Drosophila. It is not recommended for prokaryotic samples.

The one-day procedure produces directional libraries, and is Index-capable (TotalScript™ Index kit sold separately). TotalScript can be adapted to desired rRNA content and transcript coverage based on chosen priming method.

Figure 1. Overview of the TotalScript™ RNA-Seq Kit process.

Friday, April 19, 2013

Co-evolution of an RNA virus and its fungal host

Among eukaryotes with modified nuclear genetic codes, viruses are unknown. This report provides data describing an RNA virus that infects a fungal host (Scheffersomyces segobiensis), with a derived nuclear genetic code where CUG codes for serine. To determine this information, Taylor et al. extracted total RNA from yeast cells using the MasterPure Yeast RNA Purification Kit. RNA-Seq was used to sequence the putative RNA virus, examine the tRNA expression of the host, test for host expression of known fungal viral sequences, and isolate host protein-coding sequences for bioinformatics analysis. Ribosomal RNA species were removed using the Ribo-Zero Magnetic Gold kit, and library was generated using ScriptSeq™ v 2 RNA-Seq Library Preparation Kit.  RNA-Seq was performed using 50-cycle paired-end runs on two flow cells of an Illumina® HiSeq 2000. A total of 409665 reads were mapped to the totivirus with an average coverage of 4404.80 times. The putative viral sequence was confirmed by Sanger sequencing using random and specific primers for cDNA library construction.

The genomic architecture and phylogeny are consistent with infection of the fungus by a double-stranded RNA virus of the genus Totivirus. The paper further provides evidence of past or present infection with totiviruses in five species of yeasts with modified genetic codes. All but one of the CUG codons in the viral genome have been eliminated, suggesting that avoidance of the modified codon was important to viral adaptation. Authors suggest that RNA viruses co-evolved with yeasts that underwent a major evolutionary transition from the standard genetic code.

ResearchBlogging.orgTaylor, D. (2013). Virus-host co-evolution under a modified nuclear genetic code PeerJ, 1 DOI: 10.7717/peerj.50

Thursday, March 21, 2013

Ibuprofen-degrading bacterium found using CopyControl™ Fosmid Library

Disposal of pharmaceuticals into the environment through wastewater treatment systems, landfills or recycling systems is an ongoing problem that can cause serious unwanted changes in the fauna of our planet. Consequences include extinction of species, or genetic changes that can disrupt bio/ecosystems. There are microbes that can enzymatically break down many compounds and use much of it as a carbon/food source. In this report, Murdoch and Hay describe a strain of Sphingomonas that was able to at least partially degrade the anti-inflammatory/analgesic ibuprofen. Sphingomonas Ibu-2 has the unusual ability to cleave the acid side chain from the pharmaceutical ibuprofen and related arylacetic acid derivatives to yield corresponding catechols under aerobic conditions via a previously uncharacterized mechanism.

To locate the gene(s) responsible for this activity, the researchers generated a large-insert fosmid library using the CopyControl™ Fosmid Library Production Kit and screened the clones obtained by mutagenesis of the fosmids using transposon mutagenesis in standard gene knock-out experiments. Screening a chromosomal library of Ibu-2 DNA in E. coli EPI300 allowed identification of one fosmid clone that conferred the ability to metabolize ibuprofen to isobutylcatechol. Characterization of fosmid-specific loss-of-function transposon mutants permitted identification of five ORFs, ipfABDEF, whose predicted amino acid sequences bore similarity several known genes that are involved in an aromatic dioxygenase system, respectively, were also identified in a second fosmid isolated from the same library. Complementation of a markerless loss-of-function of a certain deletion mutant restored catechol production as did complementation of the Transposon mutant. Expression of subcloned ibuprofen degradation operon (ipfABDEF) alone in E. coli did not impart full metabolic activity unless it was coexpressed with the ipfHI gene. This work provides preliminary insights into the mechanism behind this novel arylacetic acid-deacylating, catechol-generating activity during ibuprofen metabolism.

ResearchBlogging.orgMurdoch RW, et al. (2013). Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2. Microbiology (Reading, England), 159 (Pt 3), 621-32 PMID: 23329679

Friday, February 22, 2013

MasterPure™ Yeast DNA used in detection of fungal meningitis

Fungal meningitis is a catastrophic infectious disease and was recently attributed to injections of contaminated methylprednisolone, which is ongoing in the U.S. The major cause of this meningitis is Exserohilum rostratum, a mold that is rarely recognized as a human pathogen. In the infectious process, the fungus has been identified from patient tissue and cerebrospinal fluid (CSF) samples, as well as unopened vials of the implicated lots of methylprednisolone, by culture or DNA sequencing, followed by a PCR assay.  Zhao et al. used the MasterPure™ Yeast DNA Purification Kit for isolation of gDNA for the development of a PCR assay to diagnose these fungal infections. The authors isolated the pathogen from patient tissue samples and CSF, cultured the fungus for 5 days on potato dextrose agar, and removed a small portion of the mycelial mat. Genomic DNA was isolated using the MasterPure Yeast DNA Purification Kit. The ability to quickly and easily isolate fungal DNA was instrumental  in developing a sensitive, rapid qPCR “Molecular beacon” assay using a specific E. rostratum probe. This assay is used for  diagnosing and tracing infected medications and can be used as a method for disease-monitoring and quality-checking the sterility of medications as well as monitoring the progress of treatment in infected patients.

ResearchBlogging.orgZhao Y, et al (2013). A Real-Time PCR Assay for Rapid Detection and Quantification of Exserohilum rostratum, a Causative Pathogen of Fungal Meningitis Associated with Injection of Contaminated Methylprednisolone. Journal of clinical microbiology, 51 (3), 1034-6 PMID: 23303500

Monday, February 18, 2013

Enrich for coding and non-coding RNAs for deeper reads into your blood RNA samples

New Product Announcement: The Globin-Zero™ Gold Kit removes the highly abundant adult globin mRNAs and the cytoplasmic and mitochondrial rRNAs from RNA isolated from human, mouse or rat blood. The rapid, single-pass process saves time and sample, and the magnetic bead protocol is suitable for high-throughput workflows.

The ScriptSeq Complete Gold Kits (Blood) generate RNA-Seq libraries from whole blood in less than 1 day. Combining the powerful Globin-Zero technology and streamlined ScriptSeq V2 library prep, the ScriptSeq Complete Kits provide a way to obtain more informative RNA-Seq as the non-globin mRNAs and large non-ribosomal, non-coding RNAs are retained. The fast and easy workflow is compatible with both intact and partially degraded blood RNA samples and is compatible with Illumina® sequencing.

Thursday, January 10, 2013

Visit Epicentre at the International PAG XXI Conference

Epicentre will be attending the International Plant and Animal Genome (PAG) XXI Conference, to be held from January 12-15 in San Diego, CA. Stop by Booth #212 to learn more about our products for RNA-Seq library preparation, plant and animal genomics, and rRNA depletion.
In addition, we will be presenting the following posters:

We hope to see you at the conference! If you're not attending, and would like more information about the products highlighted at the conference, please contact us by e-mail or call 1 (800) 284-8474 within the US.