Samstag, 29. Januar 2011

qPCR NEWS - Jan 2011 - Data Analysis & Bioinformatics & qPCR efficiency

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:
- update of new publications in qPCR Biostatistics & Bioinformatics -
- update of new publications in qPCR efficiency calculation -
- qPCR 2011 Event - Final Call for TALK and POSTER abstracts -
=> abstract submission deadline elongated till 10th Feb. 2011

Data Analysis and BioInformatics in real-time qPCR
How to do successful gene expression analysis using real-time PCR
Stefaan Derveaux, Jo Vandesompele, Jan Hellemans; Methods Vol 50, Issue 4, April 2010, in The ongoing Evolution of qPCR edited by Michael W. Pfaffl, Pages 227-230

Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. Unfortunately, what many users fail to appreciate is that numerous critical issues in the workflow need to be addressed before biologically meaningful and trustworthy conclusions can be drawn. Here, we review the entire workflow from the planning and preparation phase, over the actual real-time PCR cycling experiments to data-analysis and reporting steps. This process can be captured with the appropriate acronym PCR: plan/prepare, cycle and report. The key message is that quality assurance and quality control are essential throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, to data-analysis and finally reporting.
Further papers added =>

Determination of real-time PCR amplification efficiency
Individual samples generate different and individual fluorescence histories in kinetic RT-PCR. The shapes of amplification curves differ in the steepness of any fluorescence increase and in the absolute fluorescence levels at plateau depending on background fluorescence levels. The PCR efficiency has a major impact on the fluorescence history and the accuracy of the calculated expression result and is critically influenced by PCR reaction components
More info here =>

Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar.
Regier N & Frey B.; BMC Mol Biol. 2010 11: 57, 8 pages

BACKGROUND: RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method.
RESULTS: We quantified gene expression of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the roots of two black poplar clones, 58-861 and Poli, which were subjected to drought stress. After proving the chosen reference genes actin (ACT), elongation factor 1 (EF1) and ubiquitin (UBQ) to be constantly expressed in the different watering regimes, we applied different approaches for relative quantification to the same raw fluorescence data. The results obtained using the comparative Cq method, LinRegPCR, qBase software and the Pfaffl model showed a good correlation, whereas calculation according to the Liu and Saint method produced highly variable results. However, it has been shown that the most reliable approach for calculation of the amplification efficiency is using the mean increase in fluorescence during PCR in each individual reaction. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method.
CONCLUSIONS: As we could show that gene expression results can vary depending on the approach used for quantification, we recommend to carefully evaluate different quantification approaches before using them in studies analysing gene expression.
New added publications =>
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

qPCR 2011 Event - Final Call for TALK and POSTER abstracts -
Please submit your abstract here =>

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.
Please register here =>
Download 1st qPCR 2011 symposium announcement =>

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