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Gene Quantification Newsletter
June 2015

is sponsored by


Streamlined and automated NGS workflow

  Dear researcher,
dear Gene Quantification page reader,

Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and RT-qPCR), are compiled and summarised on the www.Gene-Quantification.info
The focus of this newsletter issue is:



Streaming portal

Join the new eConferences streaming webportal -- www.eConferences.de -- Amplify your knowledge!
This portal is dedicated to scientists from the community of qPCR, digital-PCR, NGS and Molecular Diagnostics. You’ll find here all the records from around 200 presentations held at qPCR & NGS Events in the past years. We provide the presentations via movie streaming technology in high quality - high resolution and perfect sound quality in high speed - on any internet browser or mobile device, including Android or iOS.

Impact of RNA degradation on transcript quantification

RNA-Seq -- impact of RNA degradation on transcript quantification
Gallego Romero I, Pai AA, Tung J, Gilad Y
BMC Biol. 2014 12: 42

BACKGROUND:  The use of low quality RNA samples in whole-genome gene expression profiling remains controversial. It is unclear if transcript degradation in low quality RNA samples occurs uniformly, in which case the effects of degradation can be corrected via data normalization, or whether different transcripts are degraded at different rates, potentially biasing measurements of expression levels. This concern has rendered the use of low quality RNA samples in whole-genome expression profiling problematic. Yet, low quality samples (for example, samples collected in the course of fieldwork) are at times the sole means of addressing specific questions.
RESULTS:  We sought to quantify the impact of variation in RNA quality on estimates of gene expression levels based on RNA-seq data. To do so, we collected expression data from tissue samples that were allowed to decay for varying amounts of time prior to RNA extraction. The RNA samples we collected spanned the entire range of RNA Integrity Number (RIN) values (a metric commonly used to assess RNA quality). We observed widespread effects of RNA quality on measurements of gene expression levels, as well as a slight but significant loss of library complexity in more degraded samples.
CONCLUSIONS:  While standard normalizations failed to account for the effects of degradation, we found that by explicitly controlling for the effects of RIN using a linear model framework we can correct for the majority of these effects. We conclude that in instances in which RIN and the effect of interest are not associated, this approach can help recover biologically meaningful signals in data from degraded RNA samples.

Influence of RNA extraction methods and library selection schemes on RNA-seq data
Sultan M, Amstislavskiy V, Risch T, Schuette M, Dökel S, Ralser M, Balzereit D, Lehrach H, Yaspo ML
BMC Genomics. 2014 15: 675

A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity
Adamski MG, Gumann P, Baird AE
PLoS One. 2014 9(8): e103917 -- eCollection 2014

... more papers here => RNA-integrity.Gene-Quantification.info

Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples
Zhang H, Korenková V, Sjöback R, Švec D, Björkman J, Kruhøffer M, Verderio P, Pizzamiglio S, Ciniselli CM, Wyrich R, Oelmueller U, Kubista M, Lindahl T, Lönneborg A, Rian E
PLoS One. 2014 Nov 4;9(11): e111644 -- eCollection 2014

Re-use of commercial microfluidics chips for DNA, RNA, and protein electrophoresis
Nguyen T, Kwak S, Karpowicz SJ
Biotechniques. 2014 57(5): 267-271

Long term storage of dry versus frozen RNA for next generation molecular studies
Seelenfreund E, Robinson WA, Amato CM, Tan AC, Kim J, Robinson SE
PLoS One. 2014 9(11): e111827 -- eCollection 2014

Profiling of RNA degradation for estimation of post morterm interval
Sampaio-Silva F, Magalhães T, Carvalho F, Dinis-Oliveira RJ, Silvestre R
PLoS One. 2013 8(2): e56507

Secondary and 3D structure of large RNA fragments

The mFOLD Web Server

Mfold web server for nucleic acid folding and hybridization prediction
Zuker M
Nucleic Acids Res. 2003 Jul 1;31(13):3406-15.

The abbreviated name, 'mfold web server', describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and 'energy dot plots', are available for the folding of single sequences. A variety of 'bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold

Folding and Finding RNA Secondary Structure
David H. Mathews, Walter N. Moss, and Douglas H. Turner
Cold Spring Harb Perspect Biol. 2010 Dec; 2(12): a003665.

Optimal exploitation of the expanding database of sequences requires rapid finding and folding of RNAs. Methods are reviewed that automate folding and discovery of RNAs with algorithms that couple thermodynamics with chemical mapping, NMR, and/or sequence comparison. New functional noncoding RNAs in genome sequences can be found by combining sequence comparison with the assumption that functional noncoding RNAs will have more favorable folding free energies than other RNAs. When a new RNA is discovered, experiments and sequence comparison can restrict folding space so that secondary structure can be rapidly determined with the help of predicted free energies. In turn, secondary structure restricts folding in three dimensions, which allows modeling of three-dimensional structure. An example from a domain of a retrotransposon is described. Discovery of new RNAs and their structures will provide insights into evolution, biology, and design of therapeutics. Applications to studies of evolution are also reviewed.

Sfold web server for statistical folding and rational design of nucleic acids
Ding Y, Chan CY, Lawrence CE.
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W135-41.

The Sfold web server provides user-friendly access to Sfold, a recently developed nucleic acid folding software package, via the World Wide Web (WWW). The software is based on a new statistical sampling paradigm for the prediction of RNA secondary structure. One of the main objectives of this software is to offer computational tools for the rational design of RNA-targeting nucleic acids, which include small interfering RNAs (siRNAs), antisense oligonucleotides and trans-cleaving ribozymes for gene knock-down studies. The methodology for siRNA design is based on a combination of RNA target accessibility prediction, siRNA duplex thermodynamic properties and empirical design rules. Our approach to target accessibility evaluation is an original extension of the underlying RNA folding algorithm to account for the likely existence of a population of structures for the target mRNA. In addition to the application modules Sirna, Soligo and Sribo for siRNAs, antisense oligos and ribozymes, respectively, the module Srna offers comprehensive features for statistical representation of sampled structures. Detailed output in both graphical and text formats is available for all modules. The Sfold server is available at http://sfold.wadsworth.org

3D structure of RNA fragments => RNA-integrity.Gene-Quantification.info

RNAssess -- a web server for quality assessment of RNA 3D structures
Lukasiak P, Antczak M, Ratajczak T, Szachniuk M, Popenda M, Adamiak RW, Blazewicz J
Nucleic Acids Res. 2015 Jun 11

Automated 3D structure composition for large RNAs
Popenda M, Szachniuk M, Antczak M, Purzycka KJ, Lukasiak P, Bartol N, Blazewicz J, Adamiak RW.
Nucleic Acids Res. 2012 40(14): e112

Computational Methods for RNA Structure Validation and Improvement
Jain S, Richardson DC, Richardson JS
Methods Enzymol. 2015; 558: 181-212



MIQE & qPCR iBook
How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide

1st edition published in May 2015
Editors:  Afif M. Abdel Nour & Michael W. Pfaffl
ISBN 9783000488061
Free download via iTunes --

Throughout the past 30 years Polymerase Chain Reaction (PCR) has proven to be the most powerful technique in a scientist toolbox. Only few techniques had a comparable success story like PCR. This iBook will guide you in better practicing in your laboratory thanks to the MIQE guideline.

MIQE & qPCR iBook – a digital publication: How making the MIQE guidelines easier to follow.
Afif M. Abdel Nour & Michael W. Pfaffl
Presentation at qPCR & NGS 2015 Symposium --

Please let us know, if you want to participate with an own application or chapter in the MIQE & qPCR iBook
Contact us via  iBook@bioMCC.com


20th World Congress on
Advances in Oncology


18th Intern. Symposium
Molecular Medicine


NEW Journal -- "Biomolecular Detection and Quantification" -- Call for submissions

Biomolecular Detection and Quantification (BDQ) is an open access, peer-reviewed international journal dedicated to championing excellence in molecular study design, measurement, data analysis and reporting. Its focus is on the application of qualitative and quantitative molecular methodologies to all areas of clinical and life sciences. Download the recent BDQ papers on Elsevier Science Direct.

The journal has two main aims:

  • to provide a forum for discussion and recommendation of guidelines designed to improve the accuracy of molecular measurement, its data analysis and the transparency of its subsequent reporting;
  • to publish molecular biology based studies that adhere to best practice guidelines, both current and future.

Benefits of publishing open access with Elsevier's Biomolecular Detection and Quantification - www.journals.elsevier.com/biomolecular-detection-and-quantification/

  • Peer-reviewed journal indexed by Scopus and supporting ORCID and Crossref to help maintain your publication record.
  • A renowned and respected editorial team with extensive knowledge of your research field. BDQ was established by a group of scientists based on their experience developing and publishing the Minimum Information for Publication of Quantitative Real-Time (and Digital) PCR Experiments (the MIQE and digital MIQE guidelines).
  • Reaching key audiences with 10 million active users per month using our publishing platform ScienceDirect.
  • Choice of user licenses so authors can decide how best to publish open access.
  • A discount on the publication fee for a limited time.

For more information about publishing Open Access with Elsevier, including funding body arrangements, institutional agreements and more, visit: www.elsevier.com/openaccess

We look forward to receiving your paper!

Kind regards,

The Editors
Stephen Bustin, Jim Huggett, Justin O'Grady, Michael W. Pfaffl, Carl Wittwer, Ron Cook

View full editorial board


GenEx 6:
real-time PCR data analysis software

GenEx 6 available - The most powerful tool for complex qPCR data analysis
GenEx is a popular software for qPCR data processing and analysis. 
Built in a modular fashion GenEx provides a multitude of functionalities for the qPCR community, ranging from basic data editing and management to advanced cutting-edge data analysis. Download GenEx User Guide

TATAA Biocenter, Europe´s leading provider of genomic services using quantitative real-time PCR (qPCR), and MultiD Analyses, Europe’s prime software developer for the analysis of multivariate data, release GenEx version 6 for accurate analysis of qPCR data compliant with current Clinical Laboratory Standards Institute (CLSI) guidelines.

The new analyses in GenEx 6 include: Estimating PCR efficiency, testing for outliers, testing linear model, estimating dynamic range, estimating random error, estimating limit of detection, estimating limit of quantification, estimating concentrations of unknowns, evaluation of precision, and verification of precision. For all estimates confidence intervals are calculated.
Additional new features include: Transcript distribution for single cell analysis, Survival Analysis, Receiver Operator Characteristics (ROC), Wizard for ProSeek protein analysis, and reader for 3D Gene microRNA analysis.

View our GenEx webpage and download a FREE GenEx 6 trial version => GenEx.Gene-Quantification.info

Join all GenEx talks by Prof. Mikael Kubista held at recent qPCR & NGS Events => www.eConferences.de/multid/


Best regards,

Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages



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