qPCR Newsletter - April 2012 with focus on RNA integrity -----------------------------------------------------------------------------
Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and RT-qPCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter http://qPCRnews.gene-quantification.info issue is: * RNA integrity, RNA stability, RNA decay - the page describes the importance of RNA and DNA integrity on quantitative qPCR studies - http://RNA-integrity.gene-quantification.info * Follow ou new established "GeneQuan Daily NEWS" - a daily updated newsletter - http://Daily-News.gene-quantification.info * GenEx - a powerful tool For qPCR data analysis - download a free trial version - http://GenEx.gene-quantification.info ----------------------------------------------------------------------------- RNA integrity - RNA stability - RNA decay mRNA and microRNA quality control for RT-qPCR analysis Becker C, Hammerle-Fickinger A, Riedmaier I, Pfaffl MW. Methods. 2010 50(4): 237-243 The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency. RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts (pg) and as a percentage of small RNA (%). Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity. You will find more publications on => http://RNA-integrity.gene-quantification.info -------------------------------------------------------------------------------- RNA integrity - RNA stability - RNA decay Evaluation of a novel approach for the measurement of RNA quality. Wilkes TM, Devonshire AS, Ellison SL, Foy CA. BMC Res Notes. 2010 3:89. Combined expression of miR-122a, miR-1, and miR-200b can differentiate degraded RNA samples from liver, pancreas, and stomach. Kim J, Choi NE, Oh SJ, Park SJ, Kim HK. Pathol Int. 2011 Feb;61(2): 67-72 Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements. Devonshire AS, Elaswarapu R, Foy CA. BMC Genomics. 2010 11:662. Effects of tissue handling on RNA integrity and microarray measurements from resected breast cancers. Hatzis C, Sun H, Yao H, Hubbard RE, Meric-Bernstam F, Babiera GV, Wu Y, Pusztai L, Symmans WF. J Natl Cancer Inst. 2011 Dec 21;103(24):1871-83. Epub 2011 Oct 27. Implementation of a proficiency testing for the assessment of the preanalytical phase of blood samples used for RNA based analysis. Günther K, Malentacchi F, Verderio P, Pizzamiglio S, Ciniselli CM, Tichopad A, Kubista M, Wyrich R, Pazzagli M, Gelmini S. Clin Chim Acta. 2012 Apr 11;413(7-8): 779-86 A quantitative assay for measuring mRNA decapping by splinted ligation reverse transcription polymerase chain reaction: qSL-RT-PCR. Blewett N, Coller J, Goldstrohm A. RNA. 2011 Mar;17(3): 535-543 How the RNA isolation method can affect microRNA microarray results. Podolska A, Kaczkowski B, Litman T, Fredholm M, Cirera S. Acta Biochim Pol. 2011; 58(4): 535-540 RNA Stability in Human Liver: Comparison of Different Processing Times, Temperatures and Methods. Lee SM, Schelcher C, Gashi S, Schreiber S, Thasler RM, Jauch KW, Thasler WE. Mol Biotechnol. 2012 Jan 24 Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples. Blanaid C. Mee, Paul Carroll, Simona Donatello, Elizabeth Connolly, Mairead Griffin, Barbara Dunne, Louise Burke, Richard Flavin, Hala Rizkalla, Ciara Ryan, Brian Hayes, Charles D'Adhemar, Niamh Banville, Nazia Faheem, Cian Muldoon, and Eoin F. Gaffney Biopreservation and Biobanking. December 2011, 9(4): 389-398 Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis. França A, Melo LD, Cerca N. BMC Res Notes. 2011 4:572. Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Ludyga N, Grünwald B, Azimzadeh O, Englert S, Höfler H, Tapio S, Aubele M. Virchows Arch. 2012 Feb;460(2):131-40. Epub 2012 Jan 22. Systematic discovery of structural elements governing stability of mammalian messenger RNAs. Goodarzi H, Najafabadi HS, Oikonomou P, Greco TM, Fish L, Salavati R, Cristea IM, Tavazoie S. Nature. 2012 Apr 8 An improved method for isolation of RNA from bone. Carter LE, Kilroy G, Gimble JM, Floyd ZE. BMC Biotechnol. 2012 Jan 19;12:5. Determination of RNA degradation by capillary electrophoresis with cyan light-emitted diode-induced fluorescence. Yang TH, Chang PL J Chromatogr A. 2012 Mar 30 ... ... and much more publications on => http://RNA-integrity.gene-quantification.info ----------------------------------------------------------------------------- Follow our new established "GeneQuan Daily NEWS" A daily updated newsletter around Gene Quantification and qPCR - http://Daily-News.gene-quantification.info ----------------------------------------------------------------------------- GenEx 5 - A Powerful Tool For qPCR Data Analysis Download a free trail version here => http://GenEx.gene-quantification.info 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. View our webpage => http://GenEx.gene-quantification.info Basic data editing and management Arguably the most important part of qPCR experiments is to pre-process the raw data into shape for subsequent statistical analyses. The pre-processing steps need to be performed consistently in correct order and with confidence. GenEx Standard’s streamlined and user-friendly interface ensures mistake-free data handling. Intuitive and powerful presentation tools allow professional illustrations of even the most complex experimental designs. Advanced cutting-edge data analysis When you need more advanced analyses GenEx Enterprise is the product for you. Powerful enough to demonstrate feasibility it often proves sufficient for most users demands. Current features include parametric and non-parametric statistical tests, Principal Component Analysis, and Artificial Neural Networks. New features are continuously added to GenEx with close attention to customers’ needs. New features Sample handling and samples individual biology often contribute to confounding experimental variability. By using the new nested ANOVA feature in GenEx version 5 user will be able to evaluate variance contributions from each step in the experimental procedure. With a good knowledge of the variance contributions, an appropriate distribution of experimental replicates can be selected to minimize confounding variance and maximize the power of the experimental design! For experiments with complex features, such as for example multifactorial diseases, analytical relationships and classifications may not readily be available. The support vector machine feature in the new version of GenEx is so easy to use that it will make this advanced supervised classification method easily available to novice users, while providing access to advanced parameters for experts. Download a free trail version here => http://GenEx.gene-quantification.info GenEx PDF user guides: NEW ! ! ! * GenEx user guide * GenEx user guide - Exiqon Wizard * GenEx user guide - Roche Wizard ----------------------------------------------------------------------------- Please forward this qPCR NEWS http://api.addthis.com/oexchange/0.8/forward/email/offer?url=http://qPCRnews.gene-quantification.info&title=Join+our+monthly+newsletter+on&username=qPCR-NEWS&email_template=&lng=en-us to further scientists and friends who are interested in qPCR ! Best regards, Michael W. 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