Tuesday, August 28, 2012

Whole-transcriptome RNA-Seq analysis from archival FFPE tumor samples (Part 2)

(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 to deplete rRNA from FFPE samples, enabling whole-transcriptome RNA-Seq. A recent report by Morlan et al. describes another method, called Selective Depletion of abundant RNA Species (SDRNA): the use of DNA probes homologous to human cytoplasmic and mitochondrial rRNAs.  RNA was purified using the MasterPure™ RNA Purification Kit, treated with either SDRNA or poly(A) enrichment, libraries were prepared using the Illumina® mRNA-Seq Kit or Epicentre's ScriptSeq™ mRNA-Seq Kit (now replaced by the ScriptSeq v2 Kit), and sequenced on the GAII and HiSeq 2000 instruments.

Libraries from SDRNA exhibited <1% rRNA content in uniquely mapped reads. When SDRNA libraries were compared to a poly(A)+ library, the outlier sequences were primarily identified as snRNAs, snoRNAs, scRNAs, and mirRNAs, demonstrating that a SDRNA library better represents the whole transcriptome than a poly(A)+ library. The authors mention that the Ribo-Zero Gold Kit is the only commercially available method that can effectively deplete rRNA from fragmented total RNA, and produced very comparable results to the method described in the article "with respect to the magnitude of depletion and hands-on time."

ResearchBlogging.orgMorlan JD et al. (2012). Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue. PloS one, 7 (8) PMID: 22900061

Thursday, August 23, 2012

Genome-wide comparative transcription start site analysis in enterobacteria

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 total RNA. The survey generated important information by locating transcription starts, 5'-untranslated regions, and other regulatory regions found in the transcriptome, enabling a comparison of two otherwise closely related organisms.

The technique outlined in the report is similar to that in the ExactSTART™ 5' & 3' RACE Kit. Total RNA was treated with Terminator Exonuclease to remove rRNA and degraded mRNA, enriching the sample in full-length mRNA containing 5'-triphosphorylated ends. The mRNA was treated with 5'-RNA Polyphosphatase to convert the 5'-triphosphates into 5'-monophosphates. The 5'-monophosphorylated mRNA was ligated to an adaptor molecule containing Illumina sequences using T4 RNA Ligase, and then reverse-transcribed using a modified Illumina RT primer. The resulting library was PCR-amplified and sequenced on an Illumina GAII sequencer.

The data generated were used to perform comparative genomic/gene expression studies between the two closely related organisms; the researchers identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Comparison of promoter regions and other regulatory sites revealed that 70% of the primary transcripts were expressed during exponential growth; this similarity changed dramatically when comparing TSS and regulatory elements.

The authors conclude that their comparative approach provides "a starting point for the determination of conserved and specific features of the transcriptional output of closely related bacteria at single nucleotide resolution."

ResearchBlogging.orgKim 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

Thursday, August 16, 2012

Transposon mutagenesis of Planctomyces limnophilus

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 species, the lack of molecular genetic tools has proved challenging to more in-depth study of these organisms.

Schrier et al. describe the use of the EZ-Tn5 R6Kγori/KAN-2 Transposome, co-electroporated into P. limnophilus with Epicentre's TypeOne™ Restriction Inhibitor, and successfully generated mutants in two separate experiments. Screening on kanamycin allowed the selection of transposon mutants, which were rescued using the R6Kγ origin of replication in the transposome. Recovered mutants were screened using PCR to identify those that contained an insertion within the pckA gene. The PckA gene product catalyzes the first step in the gluconeogenesis pathway; thus, a mutation in the gene's open-reading frame should result in growth inhibition in a medium not supplemented with glucose. This blockage in the mutant studied was found to be reversible, as addition of pyruvate appeared to restore wild-type growth in the absence of glucose.

The study is another example of the utility of the EZ-Tn5 Transposome system in unusual bacteria, allowing the generation and screening of mutants in specific genes to study behavior predicted from sequencing analysis.

ResearchBlogging.orgSchreier HJ et al. (2012). Transposon Mutagenesis of Planctomyces limnophilus and Analysis of a pckA Mutant. Appl Environ Microbiol PMID: 22798371

Thursday, August 9, 2012

A Tn5-based genetic screen to study tripartite class II introns

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 with the transposon at every possible position. The genetic screen allowed growth of clones only if the transposon had inserted into the group II intron such that the transcribed RNA fragments were able to assemble, trans-splice, and accurately ligate the two flanking exons. Of 201 independent clones sequenced, the Tn5 transposon was exclusively found in the sense orientation with respect to the intron, such that the terminator and promoter were functional and the intron was transcribed in two fragments. All 201 clones were unique. No clones were identified with the transposon in functionally important regions of the intron.

The study demonstrated the diverse utility of Tn5-based transposons. Further, the discovery that group II introns may be fragmented into two or three pieces and still self-assemble and splice properly provides support for an evolutionary relationship between group II introns and spliceosomal small nuclear RNAs.

ResearchBlogging.orgRitlop, 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

Monday, August 6, 2012

Ribosomal profiling to determine mechanisms of miRNA-mediated regulation in C. elegans

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 remove extraneous RNA from the ribosome preparation. Next, the poly(A) portion of the RNA was isolated from the ribosomal proteins, followed by size selection. Reverse transcription of the enriched poly(A) mRNA was performed using a novel primer that contained Illumina® sequences P5 and P7 for bridge PCR/cluster generation on a flow cell and sequencing priming sites, as well as an abasic site that allows for chemical cleavage. Circligase enzyme was used to recircularize the size-selected, single-stranded (ss) DNA. Following recircularization, the ssDNA templates were relinearized using chemical methods and PCR-enriched. The templates were then sequenced on an Illumina GAII instrument.

In this study, the authors were able to compare the relative abundance of the footprinted RNA with total mRNA isolated from the cells; they determined that functional down-regulation by some miRNAs was associated with decreases in both overall mRNA abundance and ribosome loading. They also determined that the changes were of substantially smaller magnitude than corresponding changes observed in translated protein abundance. Other miRNA targets showed only modest effects, consistent with models in which miRNA-mediated regulation occurs through a combination of mechanisms.

ResearchBlogging.orgStadler 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