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Mikael Kubista - Articles and news items
RNA levels can be measured with very high specificity, sensitivity and accuracy with techniques such as real-time quantitative PCR (qPCR), microarray analysis and next generation sequencing. This makes messenger (m) RNAs and potentially microRNAs and other non-coding RNAs popular as biomarkers. But RNA is less stable and more dynamic than DNA, and assays are not always specific for RNA, so can we trust measured expression values?
A biomarker is a biological molecule found in blood, other body fluids or tissues, and is a sign of a normal or abnormal process, or of a condition or disease1. The biomarker may be used to see how well the body responds to a treatment for a disease or condition. Most popular and common molecular biomarkers are DNA, RNA and proteins. While proteins and in particular DNA are quite stable molecules and can be analysed for many properties such as sequence years after being removed from their natural biological environment, RNA molecules are not (Table 1). The extra 2’-hydroxyl group on the ribose in RNA that is absent in DNA is a nucleophile. It confers catalytic activity to ribozymes, but also makes RNA intrinsically unstable. In aqueous solution, RNA spontaneously degrades through self-cleavage catalysed by metal ions such as Mg2+, high (>9) or low (<2) pH, and temperature. EDTA or citrate is therefore typically added to RNA preserving solutions to chelate Mg2+2. Although RNA is more resistant to ultraviolet (UV) irradiation than DNA, it causes several types of damage including photochemical modification, cross - linking and oxidation.
Setting the bar.
Q & A – Mikael Kubista from the TATAA Biocenter poses five questions for Jay Brock, Senior Manager, Applications and Technical Support, USB® Life Science Reagents from Affymetrix.
Not your grandfathers’ real-time PCR.
TATAA Biocenters, located in Gothenburg, Sweden, Prague, Czech Republic, Freising outside Münich in Germany, and Sunnyvale, California1, work with leading instrument manufacturers and reagents companies in the quantitative real-time PCR (qPCR) field on new applications, making the know-how available through hands-on courses worldwide. Every year new courses are launched based on the most recent developments in the field. The year 2008 has been very active in the qPCR area, with several important advancements that provide solid ground for future development of new research and diagnostic tools.
Issue 1 2007, Past issues / 25 January 2007 / Mikael Kubista, TATAA Biocenter and MultiD Analyses AB, Sweden, Björn Sjögreen, Center for Applied Scientific Computing, Lawrence Livermore National Laboratory, United States and MultiD Analyses AB, Amin Forootan, MultiD Analyses AB, Radek Sindelka and Jiri Jonák, Laboratory of Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic and José Manuel Andrade, Dept of Analytical Chemistry, University of A Coruna, Spain
Real-time PCR has rapidly become the preferred technique for quantitative analysis of nucleic acids. Its superior sensitivity, reproducibility and dynamic range make it the preferred choice for expression profiling in scientific, as well as routine, applications.
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