qPCR NEWS Dec 2010 - focus on RNA and DNA quality control for qPCR
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Dear researcher,
dear Gene Quantification page reader,
Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is:
- recent updates on RNA and DNA quality control - http://RNA-integrity.gene-quantification.info
- recent updates on MIQE media review - http://MIQE-press.gene-quantification.info
- qPCR 2011 Event - Call for TALK and POSTER abstracts - http://CALL.qPCR2011.net
- qPCR Application Workshops 2011 - http://site.bioeps.com/index.php?option=com_seminar&Itemid=6
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RNA quality control in miRNA expression analysis
Christiane Becker, Martina Reiter, Michael W. Pfaffl
Agilent Technologies Application Note 5990-5557EN
It is generally known that total RNA quality has a distinct influence on the validity and reliability of quantitative PCR results. In addition, the recently published MIQE guidelines focus on the pre-PCR steps and state the importance of RNA quality assessment. Various studies showed the impairing effect of ongoing RNA degradation on mRNA expression results. Therefore, the verification of RNA integrity prior to downstream applications like RT-qPCR and mircroarrays is indispensable. A fast and reliable assessment of RNA integrity can be done with the Eukaryote Total RNA Nano Assay of the Agilent 2100 Bioanalyzer. The importance of RNA quality should also be considered in new applications such as the investigation of miRNA expression profiles. With the Agilent Small RNA Assay, Agilent is offering one of the few possibilities for selectively estimating miRNA before expression analysis. However, by now little is known about factors affecting miRNA analysis. Herein, the important impact of total RNA quality on quantification of mRNA and miRNA should be considered.
http://RNA-integrity.gene-quantification.info
Newly added RNA integrity publication:
Procedures for Quality Control of RNA Samples for Use in Quantitative Reverse Transcription PCR
Tania Nolan 1,2 & Stephen Bustio 2,3
1 Sigma Aldrich,Cambridge UK; 2 Eureka Biotechnology, Cambridge UK; 3 Institute of Cell and Molecular Science, Barts and the London Queen Mary’s School of Medicine and Dentistry, London, UK
mRNA profiling in forensic genetics I - Possibilities and limitations.
Vennemann M, Koppelkamm A.
Institute of Legal Medicine, University of Freiburg, Albertstr. 9, 79104 Freiburg, Germany
Forensic Sci Int. 2010 Dec 15;203(1-3): 71-75
Postmortem mRNA profiling II - Practical considerations.
Vennemann M, Koppelkamm A.
Institute of Legal Medicine, University of Freiburg, Albertstr. 9, 79104 Freiburg, Germany
Forensic Sci Int. 2010 Dec 15;203(1-3): 76-82
Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection
Claudia Bevilacqua email, Samira Makhzami email, Jean-Christophe Helbling email, Pierre Defrenaix email and Patrice Martin email
BMC Cell Biology 2010, Published: December 2010
Robust microRNA stability in degraded RNA preparations from human tissue and cell samples.
Jung M, Schaefer A, Steiner I, Kempkensteffen C, Stephan C, Erbersdobler A, Jung K.
Department of Urology, University Hospital Charité, Berlin, Germany.
Clin Chem. 2010 Jun;56(6): 998-1006.
Assessment of mRNA and microRNA stabilization in peripheral Human Blood for Multicenter studies and Biobanks
Daniel Gilbert Weber1, Swaantje Casjens1, Peter Rozynek1, Martin Lehnert1, Sandra Zilch-Schöneweis1, Oleksandr Bryk1, Dirk Taeger1, Maria Gomolka2, Michaela Kreuzer2, Heinz Otten3, Beate Pesch1, Georg Johnen1 and Thomas Brüning11Institute for Prevention and Occupational Medicine of the German Social Accident Insuranceâ€"Institute of the Ruhr-Universität Bochum (IPA), Bochum, Germany. 2Department of Radiation Protection and Health, Federal Office for Radiation Protection, Oberschleissheim, Germany. 3German Social Accident Insurance (DGUV), Sankt Augustin, Germany.
Biomarker Insights 2010(5): 95â€"102
Pre-PCR Processing - Strategies to Generate PCR-Compatible Samples
Peter Rådström,* Rickard Knutsson, Petra Wolffs,Maria Lövenklev, and Charlotta Löfström
MOLECULAR BIOTECHNOLOGY Volume 26, 2004: 133-146
COMPARISON OF TWO AVAILABLE PLATFORMS FOR DETERMINATION OF RNA QUALITY
I. Riedmaier, M. Bergmaier and M.W. Pfaffl
Technische Universitat Munchen, Physiology Weihenstephan, Freising, Germany
Biotechnol. & Biotechnol. eq. 2010, 24(4), 2154-2159
Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA.
Okello JB, Rodriguez L, Poinar D, Bos K, Okwi AL, Bimenya GS, Sewankambo NK, Henry KR, Kuch M, Poinar HN.
Department of Anthropology, McMaster Ancient DNA Centre, McMaster University, Hamilton, Ontario, Canada.
PLoS One. 2010 Nov 10;5(11):e13931.
Stabilizing RNA at room temperature in RNAstable
Sharron Ohgi1, Laurent Coulon, Rolf Muller, Judy-Muller-Cohn, and Omoshile ClementBiomatrica, Inc., 5627 Oberlin Dr, #120, San Diego, CA 92121, USA.
Biotechniques Vol. 48 (No. 6) 2010: 470
A guide to ions and RNA structure.
Draper DE.
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA
RNA. 2004 Mar;10(3): 335-343.
Newly added DNA integrity publication:
Incorporation of measurement of DNA integrity into qPCR assays.
Brisco M, Latham S, Bartley P, Morley A.
Biotechniques. 2010 Dec;49(6): 893-897.
Department of Haematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.
Implications of storing urinary DNA from different populations for molecular analyses.
Cannas A, Kalunga G, Green C, Calvo L, Katemangwe P, Reither K, Perkins MD, Maboko L, Hoelscher M, Talbot EA, Mwaba P, Zumla AI, Girardi E, Huggett JF; TB trDNA consortium.
National Institute for Infectious Diseases L. Spallanzani, IRCCS, Roma, Italy.
PLoS One. 2009 Sep 10;4(9): e6985.
Method for isolation of PCR-ready genomic DNA from zebrafish tissues.
Meeker ND, Hutchinson SA, Ho L, Trede NS.
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.
Biotechniques. 2007 Nov;43(5):610, 612, 614.
Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repairTinaMygind*1, LarsØstergaard2, SvendBirkelund1, JesSLindholt3 and GunnaChristiansen1
1Department of Medical Microbiology and Immunology, Wilhelm Meyers Allé, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark, 2Research Unit Q, Department of Infectious Diseases, Skejby Hospital, University H
BMC Microbiology 2003, 3:19
Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues.
Okello JB, Zurek J, Devault AM, Kuch M, Okwi AL, Sewankambo NK, Bimenya GS, Poinar D, Poinar HN.
McMaster Ancient DNA Centre, Department of Anthropology, McMaster University, Hamilton, Ontario L8S4L9, Canada
Anal Biochem. 2010 May 1;400(1): 110-117
all downloads here => http://RNA-integrity.gene-quantification.info
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latest MIQE NEWS
all MIQE media and press review on http://MIQE-press.gene-quantification.info
Chapter 8 - The MIQE Guidelines Uncloaked
by Gregory L. Shipley
Publication date - January 2011
The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. This chapter presents an expanded explanation of the guideline items with commentary on how those requirements might be met prior to publication.
in PCR Troubleshooting and Optimization: The Essential Guide, ISBN: 978-1-904455-72-1
Publisher: Caister Academic Press; Editors: Suzanne Kennedy and Nick Oswald MO BIO Laboratories, Inc., Carlsbad, CA 92010, USA and BitesizeBio, Edinburgh, UK
The Marketing of Science
December 3, 2010
I am a scientist for profit. This means, as you are well aware, I have to work with marketing people to generate pretty pictures showing perfect results with any product that we sell. You know those flyers and brochures and ads in BioTechniques where a tiny picture of a gel or a qPCR assay with photoshop perfect curves or bands is plopped on the page next to some meaningless picture and supposed to convince you to call or go to a website? Those things.
Before working for a company, I would take a look at those pictures but I never put much stock into them. I mean, of course they're going to show perfect data. What else will they show? Their kit sucks next to a competitor? So marketing data never really did sway me much. I looked at it, but not in any depth. I guess, I expect there to be some attempt at science in the ad, but it's merely representative data.
My first biotech job wasn't in marketing. The company I worked for was and still is considered one of the best in the world and I was so very proud to be a part of that company. When they would introduce a new product, the product manager would come present all the beautiful R&D data proving the product works and it was convincing. I would walk away from those meetings absolutely positive that this was the best damn invention in the world and we have geniuses in R&D and how lucky am I to represent such brilliance.
About this first company, I still do believe that they have geniuses in R&D. However, since leaving, I feel that their employees are extremely self-obsessed and self-absorbed but I can understand why they are that way. It is part of the company culture. But that isn't the point of this article ......... => read more
The Story of MIQE and its Impact for Future Publications on qPCR Questions to an Expert in qPCR, Stephen Bustin (Ph.D.) - The Story of MIQE and its Impact for Future Publications on qPCR
November 2010
MIQE is a set of guidelines with some essential (59) and some desirable (28) check points for the documentation that describes the minimum information necessary for evaluation of quantitative real-time polymerase chain reaction experiments. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of quantitative PCR results. More details can be found on Stephen Bustin’s MIQE homepage: http://www.sabustin.org
Blitzlicht MIQE-Richtlinien - Qualität und Richtigkeit von qPCR-Ergebnissen steigern
Laborwelt December 2010
by Michael W. Pfaffl, TUM, Freising-Weihenstephan
Die Anwendung der quantitativen Polymerase-Kettenreaktion (qPCR) oder die Kombination der qPCR mit der reversen Transkription (RT) ist zu einem Routinewerkzeug in der modernen mole-kularbiologischen Forschung und molekularen Diagnostik geworden. Das Expression Profiling biologischer Proben auf mRNA- und microRNA-Ebene mittels quantitativer RT-PCR (RT-qPCR) ist von großem Nutzen und liefert wichtige Ergebnisse in zahlreichen biologischen Disziplinen. Vor allem in der Routinediagnostik, der universitären und industriellen Forschung sowie in der funktionellen Genomforschung ist sie unverzichtbar.
Full issue - Laborwelt - PCR Spezial - Dezember 2010
IDT publishes free downloadable qPCR user guide - User guide provides a MIQE compliant overview of this essential research technique!
8 December 2010
Integrated DNA Technologies has developed an extensive quantitative real-time polymerase chain reaction (qPCR) user guide, which is available as a free download.
The manual provides user guidance on the entire qPCR process - from RNA isolation to data analysis - covering the basics of experimental set-up, performance and analysis. Specific information on 5’ nuclease assays, including re-suspensions and qPCR protocols are also supplied, as well as a troubleshooting section which discusses commonly encountered issues. The document is written in compliance with MIQE guidelines: minimum information for publication of quantitative real-time PCR.
and much more ... ... ... http://MIQE-press.gene-quantification.info
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qPCR 2011 Event - 5th international qPCR Symposium & Industrial Exhibition & Application Workshop
28th March - 1st April 2011
in Freising-Weihenstephan, Technical University of Munich, Weihenstephan, Germany
http://www.qPCR2011.net
On behalf of the Organisation Committee and the Scientific Board it is a great pleasure to invite you to the 5th International qPCR Symposium & Industrial Exhibition & Application Workshop to be held at the Center of Life Science in Freising Weihenstephan, Technische Universität München (Germany). The great international interest in the previous meetings (qPCR 2004, qPCR 2005, qPCR 2005 in Leipzig, qPCR 2007, qPCR 2009, and qPCR 2010 in Vienna) with up to 600 participants coming from 56 countries, and over 40 international companies in the qPCR Industrial Exhibition led us to the decision to repeat the Symposium in spring 2011.
We have set the date for the qPCR 2011 Event to 28th March - 1st April 2011. The event location is the central lecture hall complex and the foyer at TUM (Technical University of Munich) in Freising Weihenstephan, Germany. The TUM and the Biotech region around Munich is part of the largest Biotech cluster in Europe, located close to the Munich airport in the heart of Bavaria.
The focus of the qPCR 2011 Event will be on: Molecular Diagnostics: from single-cells to Next Generation Sequencing
Leading academic researchers and industrial contributors in the field will participate in the symposium, which will be an arena for fruitful discussions between researchers of different backgrounds. The Symposium Talks, Poster Sessions, Industrial Exhibition and associated qPCR Application Workshops offer an overview of the present knowledge and future developments in qPCR and gene expression measurement technology and its wide applications.
The symposium will focus on 60-70 lectures and more than 100 posters will be presented by internationally recognised experts in their field. The emphasis will be on unbiased, didactic information exchange. Internationally reknown speakers will be participating in a lively and exciting programme enabling the valuable exchange of information in the qPCR field. One third of the talks will be presented by selected invited speakers, one third will be selected from the submitted abstracts and one third will be presented by qPCR related company R&D representatives. All scientific contributions will be published in the qPCR 2011 Symposium Proceedings.
Download the qPCR 2011 symposium announcement => http://www.bioeps.com/qpcr2011/qPCR-2011-event-announcement.pdf
The qPCR 2011 Event contains several parts:
* qPCR Symposium March 28th - 30th
=> talk and poster sessions
=> confirmed academic and industrial invited speakers
* A parallel qPCR Industrial Exhibition March 28th - 30th
* Followed by three qPCR application Workshops March 31st - April 1st
powered by the TATAA Biocenter & MultiD Sweden
- Basic real-time qPCR Application Workshop (2-days)
- qPCR data analysis: Biostatistics & Expression Profiling (2-days)
- MIQE guidelines (1 day) & Practical primer design (1-day)
Symposium Sessions:
- Molecular diagnostics in single-cells
- High throughput analysis in qPCR & Next Generation Sequencing
- MIQE and QM strategies in qPCR
- small RNAs qPCR - microRNA and siRNA Applications
- Digital PCR & Nano-fluidics
- Pre-analytical Steps
- qPCR BioStatistics & BioInformatics
All confirmed speakers are listed in the symposium announcement => http://www.bioeps.com/qpcr2011/qPCR-2011-event-announcement.pdf
The scientific organization is managed by international well-known scientists in the field of real-time PCR:
- Stephen Bustin, Prof. of Molecular Science QM, School of Medicine, London, UK
- Mikael Kubista, Prof. of Biotechnology, TATAA Biocenter, Sweden
- Jo Vandesompele, Prof. at the Center of Medical Genetics, University of Ghent, Belgium
- Heinrich H.D. Meyer, Prof. of Physiology, Technical University of Munich, Germany
- Michael W. Pfaffl, Reader in Physiology, TUM, Weihenstephan, Germany
scientific coordinator of the Symposium and the Application Workshops qPCR2011@wzw.tum.de
Event organization: Dr. Martina Reiter, BioEPS GmbH, Freising, Germany Martina.Reiter@BioEPS.com
We are looking forward to meeting you in March 2011 at the Symposium in Freising-Weihenstephan
Michael W. Pfaffl Martina Reiter
Symposium Chair BioEPS GmbH
Please register here => http://registration.qPCR2011.net
Please submit your abstract here => http://submission.qPCR2011.net
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BioEPS GmbH - qPCR Application Workshops
Life Science is still a growing sector and new methods and technologies are continously developed. Therefore permanent training and education becomes so important.
With our specific course program we are offering a range of high-quality course modules, in cooperation with different companies to give a general and independent overview of existing qPCR technologies and systems. Our course issues are based on skilled know-how from own research studies and publications.
Our aim is to point out a critical way of thinking to increase the quality and outcome of experimental data.
All courses are held regularly in Freising-Weihenstephan, Germany, in German and English language.
Further customized workshops and specialized trainings will be held as well across Europe and world-wide.
Workshops are powered by BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page => http://workshops.gene-quantification.info/
Course Occasions 2010:
3-day qPCR Basic Module
2-day BioStatistics & Expression Profiling Module
3-day single-cell qPCR
2-day microRNA qPCR
1-day HRM
2-das qPCR-R data analysis NEW !
Course dates 2011:
17 -19 Janurary 2011 (E) 3-day qPCR Basic Module (Mon. - Wed.)
21 - 23 February 2011 (E) 3-day qPCR Basic Module (Mon. - Wed.)
28 February - 2 March 2011 (E) 3-day single-cell qPCR Module (Mon. - Wed.)
Download course brochure => http://www.gene-quantification.de/bioeps-course-programm-2010.pdf
Register here => http://site.bioeps.com/index.php?option=com_seminar&Itemid=6
Access to our workshops => http://www.gene-quantification.de/bioeps-access.html
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Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR !
Best regards,
Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages
http://www.gene-quantification.info
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