qPCR NEWSLETTER - April 2010 - microRNA normalisation & QC
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: - microRNA quality control - microRNA normalisation - qPCR Application Workshops - and more ... ... ... European wide qPCR application workshops => register now ! Download course brochure 2010 => http://www.gene-quantification.de/bioeps-course-programm-2010.pdf Register here => http://site.bioeps.com/index.php?option=com_seminar&Itemid=6 -------------------------------------------------------------------------------- Updated section - microRNA normalisation in real-time qRT-PCR http://miRnorm.gene-quantification.info Data normalisation in microRNA experiments using qRT-PCR is a new challenge in gene quantification analysis. The reliability of any relative RT-PCR experiment can be improved by including an invariant endogenous control (reference gene) in the assay to correct for sample to sample variations in the qRT-PCR efficiency and errors in sample quantification. A biologically meaningful reporting of target mRNA copy numbers requires accurate and relevant normalisation to some standard and is strongly recommended in microRNA qRT-PCR. => But the quality of normalized quantitative expression data cannot be better than the quality of the normalizer itself. => Which are the best endogen microRNA normalizers ? => Can we apply a comparable normalising strategy as done for mRNAs ? Any variation in the normalizer will obscure real changes and produce artifactual changes. Real-time RT-PCR-specific errors in the quantification of microRNA transcripts are easily compounded with any variation in the amount of starting material between the samples, e.g. caused by sample-to-sample variation and cDNA sample loading variation. This is especially relevant when the samples have been obtained from different individuals, different tissues and different time courses, and will result in the misinterpretation of the derived expression profile of the target genes. => Therefore, normalisation of target gene expression levels must be performed to compensate intra- and inter-kinetic RT-PCR variations (sample-to-sample & run-to-run variations). Data normalisation can be carried out against an endogenous unregulated reference gene transcript or against total cellular DNA or RNA content (molecules/g total DNA/RNA and concentrations/g total DNA/ RNA). Normalisation according the total cellular RNA content is increasingly used, but little is known about the total RNA content of cells or even about the microRNA or mRNA concentrations. The content per cell or per gram tissue may vary in different tissues in vivo, in cell culture (in vitro), between individuals and under different experimental conditions. Nevertheless, it has been shown that normalisation to total cellular RNA is the least unreliable method. It requires an accurate quantification of the isolated total RNA or mRNA or microRNA fraction by optical density at 260 nm, Lab-on-Chip capillary electrophoresis instruments, or Ribogreen RNA Quantification Kit. To normalize the absolute amount according to a single reference gene (or better a set of multiple stable reference genes), further sets of kinetic PCR reactions has to be performed for the invariant endogenous control(s) on all experimental samples and the relative abundance values are calculated for internal control as well as for the target gene. For each target gene sample, the relative abundance value obtained is divided by the value derived from the control sequence in the corresponding target gene. The normalized values for different biological samples can then directly be compared. The workflow: - check for good total RNA integrity - select stable internal reference microRNA or suitable smallRNAs (via Genorm or Normfinder) - calculate reference-gene-index of selected normalizers (geometric mean of Cq) - apply relative quantification strategy (comparable to mRNA relative quantification) - apply PCR efficiency correction (if wanted) - for microRNA normalistion strategies see papers below - or find some more ideas in the Relative Quantification Section -------------------------------------------------------------------------------- some selected publications microRNA normalisation in qRT-PCR: - Overview and workflow in microRNA normalisation - Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues - Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available - A novel and universal method for microRNA RT-qPCR data normalization - Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression - A modified LOESS normalization applied to microRNA arrays: a comparative evaluation - Improved microRNA quantification in total RNA from clinical samples - Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues - Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods - Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer - High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA - Facile means for quantifying microRNA expression by real-time PCR - A single-molecule method for the quantitation of microRNA gene expression - Endogenous Controls for Real-Time Quantitation of miRNA Using TaqMan® MicroRNA Assays - microRNA normalisation in array experiments: Quality assessment and data analysis for microRNA expression arrays: - A comparison of normalization techniques for microRNA microarray data - A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA) - miChip: an array-based method for microRNA expression profiling using locked nucleic acid capture probes - A personalized microRNA microarray normalization method using a logistic regression model - Intra-platform repeatability and inter-platform comparability of microRNA microarray technology - Impact of normalization on miRNA microarray expression profiling - Comparison of normalization methods with microRNA microarray => http://miRnorm.gene-quantification.info -------------------------------------------------------------------------------- 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 BioEPS services are based on the MIQE guidelines (Minimum Information for Publication of Quantitative real-time PCR Experiments), the first guidelines for quality assurance within qPCR technology => MIQE.gene-quantification.info -------------------------------------------------------------------------------- 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 ! 1-day Project Management NEW ! 2-day Quality Management NEW ! Download course brochure 2010 => 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 -------------------------------------------------------------------------------- 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 -------------------------------------------------------------------------------- If this newsletter is not displayed correctly by your email client, please use following link: http://qPCRnews.gene-quantification.info The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright © 2005 - 2010 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newslet...@gene- quantification.info?subject=SUBSCRIBE _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
