Freitag, 17. Oktober 2014

HOT PAPER -- miRQC study -- microRNA quality control study

HOT PAPER:
Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study
Mestdagh P, Hartmann N, Baeriswyl L, Andreasen D, Bernard N, Chen C, Cheo D, D'Andrade P, DeMayo M, Dennis L, Derveaux S, Feng Y, Fulmer-Smentek S, Gerstmayer B, Gouffon J, Grimley C, Lader E, Lee KY, Luo S, Mouritzen P, Narayanan A, Patel S, Peiffer S, Rüberg S, Schroth G, Schuster D, Shaffer JM, Shelton EJ, Silveria 9, Ulmanella U, Veeramachaneni V, Staedtler F, Peters T, Guettouche T, Vandesompele J
Nature Methods 11, 809–815 (2014)

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.





Dienstag, 14. Oktober 2014

PCR and qPCR Webinar Series -- A six webinar series on PCR techniques

PCR and qPCR Webinar Series -- A six webinar series on PCR techniques

This six webinar series on PCR will be based on MIQE guidelines. MIQE, Minimum Information for Publication of Quantitative-Real Time PCR, helps scientists to design and report reliable and reproducible qPCR results. They help others to understand and replicate already conducted experiments. In this webinar series we will discuss step by step critical points which need to be taken into consideration in order to acquire reliable data. 

Topics will include:
- Basic principles of  PCR, qPCR and ddPCR
- qPCR Assay Design
- Importance of Sample Quality in qPCR Analysis
- Improving Assay Quality by Optimization and Validation
- Data Analysis
- Troubleshooting

- See more at: http://www.sigmaaldrich.com/life-science/learning-center/customer-education/pcr-webinar-series.html

 Speakers

Marina Wiklander, PhD
Application Specialist, Sigma Custom Products
Marina received her BSc at Tartu University in Estonia at the Department of Cell and Molecular Biology. In 2001 she moved to Sweden and started her MSc in Molecular Genetics at Department of Plant Biology in Swedish University of Agricultural Sciences. Marina completed her PhD at Department of Genetics and Pathology in Rudbeck Laboratory at Uppsala University. Her thesis was dealing with epigenetic changes of transcription factor genes in haematopoietic tumours. For the last 5 years working as an application specialist in the technical and application support team, providing customers with: assay designs and evaluations, seminars and workshops.

Anders Bergkvist, PhD
Application Specialist, Sigma Custom products
Dr. Bergkvist has a strong multidisciplinary academic and industry background with a M.Sc  on Engineering Physics from Chalmers University of Technology and a Ph.D. in Biochemistry from Goteborg University in Goteborg and a postdoc in Bioinformatics from Harvard Medical School in Boston. The common theme of his work has been applications of computational tools to biologically relevant tasks. At present he is providing customers with design support and developing new business and marketing opportunities as an Application Specialist at Sigma Life Science.
Tania Nolan, PhD
The Gene Team-Consultant for Sigma-Aldrich
Tania Nolan is founder and CEO of The Gene Team Ltd; an international consortium of expert Life Scientists who provide educational and project support to all researchers. She has an international reputation for expertise in the field of mRNA quantification using RT-qPCR and much more importantly, for being able to troubleshooting absolutely any problem! She edited PCR Technologies: Current Innovations with Stephen Bustin and was a significant co-author of the specialist textbook “The A - Z of Quantitative PCR” (ed S.A. Bustin; IUL Press). Tania is an enthusiastic lecturer and has presented several plenary lectures and chaired sessions at major international meetings. Teaching is a particular passion for Tania and she regularly organizes qPCR workshops worldwide. She has an active publication record and regularly contributes to the scientific literature, mainly addressing aspects of quality control of qPCR and qRT-PCR experiments. Tania gained a first class honors degree and was awarded the Excellence in Research Prize from undergraduate studies (University of Salford) and then a PhD in genetics from Manchester University, UK. She took up an AstraZeneca Research Fellowship to study the genetic regulation in breast cancer before moving to Stratagene to support the launch of their qPCR program. Tania lead the Sigma Aldrich Custom Products technical and application support team and was awarded an Honorary Senior Lecturer position at Manchester University in 2014. Outside of science, Tania is a keen clarinettist and plays in a local orchestra as well as small ensemble groups. While on lecture tours, she travels with her clarinets and practices whenever possible.

Donnerstag, 9. Oktober 2014

The Top 10 Most Cited Articles in Clinical Chemistry

The Top 10 Most Cited Articles in Clinical Chemistry

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments
Authors: Bustin, Stephen A.; Benes, Vladimir; Garson, Jeremy A.; et al.
Volume 55 Issue 4: April 2009
http://www.clinchem.org/content/59/3/581.full?sid=b34e33e7-61d6-43f4-8bbf-8da7d95bb511

... more info  MIQE.gene-quantification.info

Dienstag, 30. September 2014

Bio-Rad’s Universal Real-Time PCR App – Everything You Need to Know about Real-Time PCR at Your Fingertips

Bio-Rad’s Universal Real-Time PCR App – Everything You Need to Know about Real-Time PCR at Your Fingertips

Quickly find everything you need to know about real-time PCR at your fingertips with our Universal Real-Time PCR app. Download today!  http://bit.ly/1ttIemF

Bio-Rad’s Universal Real-Time PCR Reagents App brings you in-depth product information for all of our one-step and two-step qPCR kits and reagents. These reagents utilize our proprietary blend of passive reference dyes that enables their use on all real-time PCR systems.
Features of the app include:
  • Individual and competitive performance data compared to other leading products
  • Interactive tutorials covering the qPCR basics on how reverse transcription works and why not all cDNA synthesis kits are created equal
  • Technical manuals
  • Product inserts
  • Troubleshooting guides
  • And more ... ... 

Donnerstag, 18. September 2014

Optimization of Extraction of Circulating RNAs from Plasma - Enabling Small RNA Sequencing.


Optimization of Extraction of Circulating RNAs from Plasma - Enabling Small RNA Sequencing.
Spornraft M, Kirchner B, Haase B, Benes V, Pfaffl MW, Riedmaier I
PLoS One. 2014 9(9): e107259 - eCollection 2014

There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the state-of-the-art technology in massive parallel sequencing that enables a comprehensive characterization of the whole transcriptome. Screening the transcriptome for biomarker signatures accelerates progress in biomarker profiling for molecular diagnostics, early disease detection or food safety. Therefore, the aim was to optimize a method that enables the extraction of sufficient amounts of total RNA from bovine plasma to generate good-quality small RNA Sequencing (small RNA-Seq) data. An increased volume of plasma (9 ml) was processed using the Qiagen miRNeasy Serum/Plasma Kit in combination with the QIAvac24 Plus system, a vacuum manifold that enables handling of high volumes during RNA isolation. 35 ng of total RNA were passed on to cDNA library preparation followed by small RNA high-throughput sequencing analysis on the Illumina HiSeq2000 platform. Raw sequencing reads were processed by a data analysis pipeline using different free software solutions. Seq-data was trimmed, quality checked, gradually selected for miRNAs/piRNAs and aligned to small RNA reference annotation indexes. Mapping to human reference indexes resulted in 4.8±2.8% of mature miRNAs and 1.4±0.8% of piRNAs and of 5.0±2.9% of mature miRNAs for bos taurus.