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.

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

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."

Sahl, 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

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.

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:

• #10: Rapid and Efficient Methods for Preparing rRNA-Depleted and Directional Plant RNA-Seq Libraries
• #793: An Improved rRNA Removal Process for RNA-Seq Library Preparation

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.

Large-insert cloning aids study of dinoflagellate species

Large-insert cloning products have long been a mainstay of Epicentre's product line and are critical for the study of gene expression and interactions. Recently, Jaeckisch et al. described making three kinds of libraries (cDNA, fosmid, and BAC) for characterizing the marine dinoflagellate Alexandrium ostenfeldii.

Many dinoflagellate species are notorious for the toxins they produce, as well as ecological and human health consequences associated with harmful algal blooms (HABs). One way to study these otherwise toxic compounds is to build their synthesis genes into a large-insert-capable, low-copy cloning vector, transform into a suitable host cell, and then perform the desired studies in such a way that the genes will not have a negative affect on the host. The genes for the toxins cited in the publication (macrocyclic imine toxins, described as spirolides), were inserted into the pCC1FOS vector and transfected into the TransforMAX™ EPI300 host strain for further study. Further, genomic DNA from A. ostefendii was prepared for cloning into the pIndigoBAC-5 HindIII Cloning-Ready vector and transformation into the TransforMAX EC100 host.

A total of 384 BAC clones were obtained with insert sizes ranging from 50 to 150 kb, which provided sufficient coverage to allow elucidation of the whole genome sequence and some comparative sequence data with the marine dinoflagellate H. triquetra. The authors used the sequence information obtained from the BAC and CopyControl fosmid libraries to investigate spliced leader (SL) trans-splicing and mRNA transposition mechanisms. They characterized the genome using selected clones, using a combination of transcriptomic data and random genomic clones. Examination of SL sequences revealed similar features as in other dinoflagellates, including other Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. The transposition observed in these genes probably contributes to overall genome complexity by generating additional gene copies.
The authors conclude:

The genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date...The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally [sic] genome organization of dinoflagellates with a low amount of histones and histone-like proteins.

Jaeckisch, N. et al. (2011). Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii PLoS ONE, 6 (12) DOI: 10.1371/journal.pone.0028012

What's new in the ScriptSeq v2 kit?

Recently, Epicentre launched the ScriptSeq™ v2 RNA-Seq Kit for preparing directional, ligation-free RNA-Seq libraries in 4 hours. This kit offers several advantages over the previous version:

• Transcript coverage is improved and GC bias is reduced by an improved terminal-tagging oligo (TTO).
• Less input RNA required: Libaries can be prepared from as little as 500 pg of rRNA-depleted or poly(A)-enriched RNA.
• A streamlined protocol and premixed reagents make the kit easier to use and require fewer pipetting steps.
• A signifcantly lower price compared to the original kit.

The original ScriptSeq Kit will be available for a transition period, to allow existing customers to finish important projects. We expect customers will find that the new ScriptSeq v2 Kit provides better performance at a lower price.

Role of Piwi proteins in mammalian transposon silencing

In a recent Nature publication, Reuter et al. report on the study of certain small RNAs that affect the fertility of male mice. Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi (also known as Piwil4) in a genome defense mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility.

The researchers cite the use of Epicentre’s Ribo-Zero™ Kit (Human/Mouse/Rat) to remove interfering ribosomal RNA from the experimental matrix, allowing closer study of the interactions between piRNAs. They also used the ScriptSeq™ mRNA-Seq Kit for an unusual application: sequencing small RNAs. Under normal circumstances, small RNAs <50-60 nucleotides are better suited for Epicentre's ScriptMiner™ Small RNA-Seq Kit, due to potential issues with the ScriptSeq method at the 5’ end of small RNAs. These results demonstrate that the ScriptSeq Kit has the ability to prepare RNA-Seq libraries from RNA types other than mRNA, including small RNAs.

Reuter, M. et al. (2011). Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing Nature, 480 (7376), 264-267 DOI: 10.1038/nature10672