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.

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.

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

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.

Murdoch 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

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.

Zhao 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

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.

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:

• P0022: An innovative solution to RNA-Seq sample prep from high quality RNA
• P0026: A rapid end-to-end workflow for preparing rRNA-depleted and directional plant RNA-Seq libraries

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.

New Product Announcement: ARTseq™ Ribosome Profiling Kits

ARTseq™ Ribosome Profiling Kits provide a ‘snapshot’ of the ribosomes active in a cell at a specific time point by sequencing only the ribosome-protected mRNA fragments. This powerful method can be used to investigate translational control, measure gene expression, and predict protein abundance. ARTseq is available for mammalian and yeast cell lines.