Monday, January 30, 2012

Transcriptome-wide discovery of circular RNAs in Archaea

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 permuted mapping to the genome, a characteristic of circular RNA. They developed a workflow to enrich for circular transcripts and overcome possible artifacts, by pretreating the RNA sample using RNase R. The isolated circular RNA was used in the development of a new sequencing method, "circRNA-Seq", which uses enriched circular RNAs and allows quantification of relative abundance/prevalence of these RNAs in the cell in an unbiased way. The authors applied the technique to the archaeon Sulfolobus solfataricus P2. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but also many noncoding RNAs and circular RNAs of unknown function. Many of the identified circles were conserved in S. acidocaldarius, further supporting their functional significance. The data suggest that circular RNAs, especially circular noncoding RNAs, are more common in archaea than previously recognized. The circRNA-seq method will enable the study of these novel RNAs in any organism and will help to determine their relative importance in the biology of the cell.

ResearchBlogging.orgDanan, M. et al. (2011). Transcriptome-wide discovery of circular RNAs in Archaea Nucleic Acids Research DOI: 10.1093/nar/gkr1009

Wednesday, January 25, 2012

Transcriptional profiling of an enterotoxigenic E. coli isolate

Ribo-Zero Kits have rapidly established themselves as the method of choice for depleting ribosomal RNA (rRNA) for RNA-Seq studies. Their utility extends to microarray-based analysis of gene expression. Sahl et al. describe the use of RNA-Seq and microarray analysis to study the effects of chemical signaling factors in the pathogenesis of enterotoxigenic Escherichia coli (ETEC). This organism is an important pathogenic variant (pathovar) of E. coli in developing countries and is associated with significant morbidity and mortality rates.

The research focused on providing various stimulants to the cells in culture followed by RNA extraction to measure the up- or down-regulation of the enterotoxins being produced by the cells. The Ribo-Zero Gram-Negative Kit was used to remove rRNA from the total RNA preparations, simplifying data analysis from RNA-Seq libraries and microarray expression analysis. RNA-Seq results demonstrated bile salts regulate many virulence factors; one of the most differentially expressed genes in the presence of bile is a unique plasmid-encoded AraC-like transcriptional regulator (peaR).

The authors conclude: "These results provide transcriptional targets and putative mechanisms to better understand the global regulatory networks and virulence expression in this important human pathogen."

ResearchBlogging.orgSahl, J. and Rasko, D. (2012). Analysis of the global transcriptional profiles of enterotoxigenic Escherichia coli (ETEC) isolate E24377A Infection and Immunity DOI: 10.1128/IAI.06138-11

Thursday, January 12, 2012

Genomic study of deadly bovine pathogen faciliated by QuickExtract Bacterial Kit

Mannheimia haemolytica is a Gram-negative bacterium associated with bovine respiratory disease complex. During stress, such as a viral infection and/or transportation to the feedlot, the bacterium transforms from benign to deadly, and causes a disease called shipping fever, resulting in losses of more than $1 billion annually in the U.S. Despite its economic importance, there are no specific and accurate genetic markers for this disease.

Researchers at Washington State University (Lawrence PK et al., BMC Genomics 2010, 11:535) performed a three-way comparison between the genomic sequences of three strains of M. haemolytica from cattle and domestic sheep. They extracted total genomic DNA using the QuickExtract Bacterial DNA Extraction Kit, and prepared genomic libraries for sequencing on a Genome Sequencer FLX (Roche). At 20X sequence coverage, the authors identified a number of genes that are unique to each strain. In addition, many high-confidence single nucleotide polymorphisms (hcSNPs) were identified, which will be used to design new arrays to study variation across strains and potentially aid in understanding gene regulation and mode of action. Additional virulence factors included a previously unknown type III secretion system, and CRISPR loci that indicates the potential resistance of M. haemolytica to superinfection by phages. The study also identified various adhesins, containing protein cleavage domains, that could potentially serve as effective vaccine targets.

Friday, January 6, 2012

Visit Epicentre at the International PAG XX Conference

Epicentre will be attending the International Plant and Animal Genome (PAG) XX Conference, to be held from January 14-18 in San Diego, CA. Stop by Booth #209 to learn more about our products for RNA-Seq library preparation, plant and animal genomics, and to receive special discounts on a variety of Epicentre products.

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.