Dienstag, 13. Oktober 2015

BDQ Special Issue: Advanced Molecular Diagnostics for Biomarker Discovery – Part I

Biomolecular Detection and Quantification
Volume 5, Pages 1-38 (September 2015) 
Special Issue: Advanced Molecular Diagnostics for Biomarker Discovery – Part I
Edited by Michael W. Pfaffl


  • Guest editor's introduction for BDQ special issue: ‘Advanced Molecular Diagnostics for Biomarker Discovery’
    Pages 1-2
    Michael W. Pfaffl
  • Effects of post-mortem and physical degradation on RNA integrity and quality.
    Pages 3-9
    Monika Sidova, Silvie Tomankova, Pavel Abaffy, Mikael Kubista, Radek Sindelka
  • Removal of between-run variation in a multi-plate qPCR experiment.
    Pages 10-14
    Jan M. Ruijter, Adrián Ruiz Villalba, Jan Hellemans, Andreas Untergasser, Maurice J.B. van den Hoff
  • The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics—Identifying biomarker signatures by multivariate data analysis.
    Pages 15-22
    Spornraft Melanie, Kirchner Benedikt, Michael W. Pfaffl, Riedmaier Irmgard
  • Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.
    Pages 23-29
    Francesca Salvianti, Giada Rotunno, Francesca Galardi, Francesca De Luca, Marta Pestrin, Alessandro Maria Vannucchi, Angelo Di Leo, Mario Pazzagli, Pamela Pinzani
  • Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing.
    Pages 30-37
    Thomas Blomquist, Erin L. Crawford, Jiyoun Yeo, Xiaolu Zhang, James C. Willey

Montag, 21. September 2015

The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics

The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics — Identifying biomarker signatures by multivariate data analysis
in Biomolecular Detection and Quantification
Spornraft Melanie, , Kirchner Benedikt , Michael W. Pfaffl , Riedmaier Irmgard 

Worldwide growth and performance-enhancing substances are used in cattle husbandry to increase productivity. In certain countries however e.g., in the EU, these practices are forbidden to prevent the consumers from potential health risks of substance residues in food. To maximize economic profit, ‘black sheep‘ among farmers might circumvent the detection methods used in routine controls, which highlights the need for an innovative and reliable detection method. Transcriptomics is a promising new approach in the discovery of veterinary medicine biomarkers and also a missing puzzle piece, as up to date, metabolomics and proteomics are paramount. Due to increased stability and easy sampling, circulating extracellular small RNAs (smexRNAs) in bovine plasma were small RNA-sequenced and their potential to serve as biomarker candidates was evaluated using multivariate data analysis tools.
After running the data evaluation pipeline, the proportion of miRNAs (microRNAs) and piRNAs (PIWI-interacting small non-coding RNAs) on the total sequenced reads was calculated. Additionally, top 10 signatures were compared which revealed that the readcount data sets were highly affected by the most abundant miRNA and piRNA profiles. To evaluate the discriminative power of multivariate data analyses to identify animals after veterinary drug application on the basis of smexRNAs, OPLS-DA was performed. In summary, the quality of miRNA models using all mapped reads for both treatment groups (animals treated with steroid hormones or the β-agonist clenbuterol) is predominant to those generated with combined data sets or piRNAs alone. Using multivariate projection methodologies like OPLS-DA have proven the best potential to generate discriminative miRNA models, supported by small RNA-Seq data. Based on the presented comparative OPLS-DA, miRNAs are the favorable smexRNA biomarker candidates in the research field of veterinary drug abuse.

Freitag, 18. September 2015

qPCR NEWS Sept. 2015 -- Molecular Regulatory Networks

Our Sept. 2015 newsletter -- qPCRnews.Gene-Quantification.info -- informs about the latest news in quantitative real-time PCR (qPCR and RT-qPCR), are compiled and summarised on www.Gene-Quantification.info

The focus of 09/2015 newsletter issue is:

Freitag, 14. August 2015

Integrative Analysis of MicroRNA and mRNA Data Reveals an Orchestrated Function of MicroRNAs in Skeletal Myocyte Differentiation in Response to TNF-α or IGF1

Integrative Analysis of MicroRNA and mRNA Data Reveals an Orchestrated Function of MicroRNAs in Skeletal Myocyte Differentiation in Response to TNF-α or IGF1


Skeletal muscle cell differentiation is impaired by elevated levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α) with pathological significance in chronic diseases or inherited muscle disorders. Insulin like growth factor-1 (IGF1) positively regulates muscle cell differentiation. Both, TNF-α and IGF1 affect gene and microRNA (miRNA) expression in this process. However, computational prediction of miRNA-mRNA relations is challenged by false positives and targets which might be irrelevant in the respective cellular transcriptome context. Thus, this study is focused on functional information about miRNA affected target transcripts by integrating miRNA and mRNA expression profiling data.

Methodology/Principal Findings

Murine skeletal myocytes PMI28 were differentiated for 24 hours with concomitant TNF-α or IGF1 treatment. Both, mRNA and miRNA expression profiling was performed. The data-driven integration of target prediction and paired mRNA/miRNA expression profiling data revealed that i) the quantity of predicted miRNA-mRNA relations was reduced, ii) miRNA targets with a function in cell cycle and axon guidance were enriched, iii) differential regulation of anti-differentiation miR-155-5p and miR-29b-3p as well as pro-differentiation miR-335-3p, miR-335-5p, miR-322-3p, and miR-322-5p seemed to be of primary importance during skeletal myoblast differentiation compared to the other miRNAs, iv) the abundance of targets and affected biological processes was miRNA specific, and v) subsets of miRNAs may collectively regulate gene expression.


Joint analysis of mRNA and miRNA profiling data increased the process-specificity and quality of predicted relations by statistically selecting miRNA-target interactions. Moreover, this study revealed miRNA-specific predominant biological implications in skeletal muscle cell differentiation and in response to TNF-α or IGF1 treatment. Furthermore, myoblast differentiation-associated miRNAs are suggested to collectively regulate gene clusters and targets associated with enriched specific gene ontology terms or pathways. Predicted miRNA functions of this study provide novel insights into defective regulation at the transcriptomic level during myocyte proliferation and differentiation due to inflammatory stimuli.

Donnerstag, 13. August 2015

Exosome and microRNA transfer from paracrine & endocrine to cross-kingdom communication

Exosome and microRNA transfer
from paracrine & endocrine to cross-kingdom communication

In multicellular organisms, the cell-to-cell communication is of particular importance for any physiological process, and the proper organization of the entire organism. Numerous studies over the past decade suggest a horizontal transfer of cellular secreted microRNAs between cells, tissues and organs. Hence extracellular RNAs (primarily small non-coding RNAs) represent a novel form of inter-cellular communication by transferring genetic information from a donor cell to a recipient cell. This points to an important new role for small RNAs in inter-cellular communication on the paracrine- and endocrine-level.

Para- and endocrine communication
Small RNAs (primary microRNAs, piRNAs and other small RNA families) can be exported out of the donor cells and transported by various carriers, e.g. membrane-derived vesicles (exosomes, microvesicles, ectosomes, apoptotic bodies, and more), microRNA-binding protein complexes (RBP), or high density lipoproteins (HDL). Secreted microRNAs can be delivered into recipient cells where they function as endogenous microRNAs, simultaneously regulating multiple target genes or signaling pathways.

Cross-kingdom communication
In prokaryotes, this molecular signaling is typically referred to as quorum sensing, whereas in eukaryotic cells, the molecular communication occurs through hormones and cytokines. Recently various publications report that microRNAs can also be transmitted from one species to another, inducing signal interference in distant species, even in a cross-kingdom manner. This new mode of cross-species communication might mediate symbiotic and pathogenic relationships between various organisms. 
This can be of enormous importance in the inter-species communication of microorganisms and their hosts or by diet-derived small RNAs. Higher organisms are constantly under attack from pathogens, resulting in severe consequences on global human or veterinary health. Hence small RNA mediated RNA interference (RNAi) is a conserved regulatory mechanism that is involved in almost all eukaryotic cellular processes, including host immunity and pathogen virulence. Recent evidence supports the significant contribution of small RNAs and RNAi to the communication between hosts and some eukaryotic pathogens or symbiotic microorganisms. Mobile silencing signals - most likely small RNAs - are capable of translocating from the host to its interacting organism, and vice versa.