We show that assessment of ctDNA is a non-invasive, exquisitely specific and highly sensitive approach for monitoring disease load, which has the potential to provide clinically relevant lead times compared with conventional methods. Furthermore, we provide a low-coverage protocol optimised for identifying SSVs with excellent correlation between SSVs identified in tumours and matched metastases. Application of ctDNA analysis has the potential to change clinical practice in the management of CRC.

Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional elongation. Indeed, we find that a pool of heat shock HSP104 transcripts are 3-end truncated in THO complex mutant as well as sub2 mutant backgrounds. Surprisingly, however, this defect can be suppressed by deletion of the 3-5 exonuclease Rrp6p. This indicates that incomplete RNAs result from nuclear degradation rather than from a failure to efficiently elongate transcription. RNAs that are not degraded are retained at the transcription site in a Rrp6p-dependent manner. Interestingly, the addition of a RRP6 deletion to sub2 or to THO complex mutants shows a strong synthetic growth phenotype, suggesting that the failure to retain and/or degrade defective mRNAs is deleterious. mRNAs produced in the 3-end processing mutants rna14-3 and rna15-2, as well as an RNA harboring a 3 end generated by a self-cleaving hammerhead ribozyme, are also retained in Rrp6p-dependent transcription site foci. Taken together, our results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p.The physical separation of mRNA biogenesis and bulk protein synthesis in eukaryotic cells necessitates molecular transport between the nuclear and cytoplasmic compartments. To form a mature translatable mRNA, several processing steps must be completed accurately in the nucleus. In successful formation of mature translatable mRNAs, the 5Ј end of the transcript acquires a methylated guanosine cap structure, introns are excised, and the 3Ј end is cleaved and polyadenylated (for a review, see reference 31). These pre-mRNA modifications serve to protect the transcript from the cellular degradation systems as well as prepare it for downstream events. Over the past few years, it has been established that pre-mRNA processing is carefully coordinated with transcription, and processing events are thought to occur mostly while the premRNA is nascent, i.e., still emerging from the transcription machinery (29). Nuclear mRNA export is tightly linked to pre-mRNA processing and, consequently, probably also to transcription. In metazoans, although not strictly required for export, pre-mRNA splicing has been found to make loading of export factors onto the mRNA more efficient: the exon junction complex associates with the mRNA as a consequence of splicing, and the exon junction complex-associated proteins Aly/REF and UAP56 are important for productive mRNA export (14,22,24,25,28,32). The Saccharomyces cerevisiae orthologues of these two proteins (the RNA binding protein Yra1p and the DECD-box RNA helicase Sub2p, respectively) are also involved in mRNA export in this organism (18,37,38,40). Aly/Yra1p is b...

The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3 0 -5 0 exonuclease Rrp6p is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein is fully active, suggesting polyadenylation-independent mRNA degradation. Transcription pulse-chase experiments show that HSP104 molecules undergoing quality control in THO/sub2 mutant strains fall into two distinct populations: One that is quickly degraded after transcription induction and another that escapes rapid decay and accumulates in foci associated with the HSP104 transcription site.

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A) + RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.

Supervision of graduate students is a core activity in higher education. Previous research on graduate supervision focuses on individual and relational aspects of the supervisory relationship rather than collective, pedagogical and methodological aspects of the supervision process. In presenting a collective model we have developed for academic supervision of Danish master students, we seek to fill these gaps. The underlying pedagogical rationale for the model is that students' participation and learning are interconnected. The model provides possibilities for incorporating a progressive and systematic interaction between master students in their individual writing processes. In the article, we investigate the potentials and challenges of the model and draw on analyses of six individual interviews with master students and one focus group interview with five supervisors. Our findings show that students learn core academic competencies in collective academic supervision (CAS), such as the ability to assess theoretical and practical problems in their practice and present them to peers. The analysis reveals that interaction between divergent projects and voices in CAS can be highly productive in academic learning. However, the model also challenges both students and supervisors because both parties are used to a oneto-one supervisory relationship and not prepared for different modes of participation and learning. According to both supervisors and students, the majority of supervisors need better training in the facilitation of collective supervision processes.

A common method for calculating results from qPCR experiments is the comparative Ct method, also called the 2(-ΔΔCt) method. However, several assumptions are included in the 2(-ΔΔCt) method and standard statistical analyses are not directly applicable. Here, we describe a different method, the X(0) method, for result calculations and statistical analysis from qPCR experiments. The X(0) method differs from the 2(-ΔΔCt) method by introducing a conversion of the exponentially related Ct values into linearly related X(0) values, which represent the amount of starting material in a qPCR experiment. Results calculated by the X(0) method are illustrated for qPCR experiments with technical and biological replicates, including procedures to calculate standard deviations. Incorporation of primer efficiencies in calculations by the X(0) method is also described. Altogether, the X(0) method constitutes a very simple and accurate alternative to the 2(-ΔΔCt) method for result calculations from qPCR data.

In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal site of transcription, and known S. cerevisiae nuclear structures such as the nucleolus and the nucleolar body. Our results show that retained SSA4 RNA localizes to an area in close proximity to the SSA4 locus. On deletion of Rrp6p and release from the genomic locus, heat shock mRNAs produced in the rat7-1 strain colocalize predominantly with nucleolar antigens. Bulk poly(A) + RNA, on the other hand, is localized primarily to the nuclear rim. Interestingly, the RNA binding nucleocytoplasmic shuttle protein Npl3p shows strong colocalization with bulk poly(A) + RNA, regardless of its nuclear location. Taken together, our data show that retention occurs close to the gene and indicate distinct nuclear fates of different mRNAs.

The Boyden chamber assay has been developed for various cell migration and invasion protocols. One variant of the Boyden chamber assay is the pseudopodium isolation assay, which has been developed to identify RNA and proteins localized in pseudopodia cell protrusions. Astrocytes are the most abundant cell type in the CNS and typically extend long cellular protrusions. Increasing interest emerges concerning for example the growth mechanisms and functions of astrocytes in respect to brain development, re-uptake of neurotransmitters in the synaptic cleft and glial scar formation. Protein and RNA localization mechanisms have been extensively examined in neurons and shown to play pivotal roles for the functional presence of specific protein components in neuronal protrusions. Here we present a simple Boyden chamber based method to isolate astrocyte cell protrusions for biochemical analysis. We have succeeded in isolating protrusion localized RNA and protein from the mouse astrocyte cell line, C8-S, and mouse primary astrocytes. This is exemplified in biochemical analyses showing specific localization of the mRNA for Ras-related protein (Rab13), Plakophilin-4 (Pkp4), Ankyrin Repeat Domain 25 (Ankrd25), and inositol polyphosphate-1-phosphatase (Inpp1) in the protrusions of both C8-S cells and primary astrocytes. Concordant, the Pkp4 protein was also predominantly localized in the protrusions of C8-S and primary astrocytes. The described methodology can be the basis for both genome wide and specific descriptive and functional studies of RNA and protein localization in the protrusions of astrocytes which could contribute considerably to the existing knowledge of astrocyte functions in the CNS.