  RNA
integrity (1)
OVERVIEW
REVIEW: RNA integrity and the effect on the real-time qRT-PCR performance. Molecular Aspects of Medicine 27 (2006) 126–139 Simone Fleige & Michael W. Pfaffl Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL), Technical University of Munich, 85350 Freising, Germany TATAA Biocenter Germany, Freising-Weihenstephan, Germany The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern molecular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as good total RNA quality and higher than eight as perfect total RNA for downstream application. Correcting
false gene expression measurements from degraded RNA using RT-qPCR.
Matthias Port, Hans Ulrich Schmelz, Tanja Stassen, Kerstin Mueller, Marcus Stockinger, Richard Obermair & Michael Abend Bundeswehr Institute of Radiobiology, Munich; Department of Hematology and Oncology, Hannover Medical Department of Urology, Federal Armed Forces Hospital, Koblenz; Institute of Veterinary Pathology, LMU, Munich, Germany. Diagn Mol Pathol 2007;16:38–49 This paper describes a method allowing correcting false gene expression measured on highly degraded RNA using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). RNA was isolated from different models (in vitro cell lines, in vivo models of human and dog) and different tissue types. In vitro RNA degradation and modeling of in vivo degradation were applied on intact and degraded total RNA. Gene expression (eg, Bcl-2, GAPDH, PGK, PSME3, RAB2, BAX) was measured using RT-qPCR. 18S rRNA proved to be the most constant house-keeping gene. Less than 10-fold degraded RNA can be quantified correctly when using 18S rRNA for normalization purposes. Higher-fold degraded RNA can be quantified correctly up to a precision that is comparable to RTQ-PCR measurements on intact RNA when simulating the RNA-species and tissue-specific degradation kinetic. Einfluss
der RNA Integrit auf die quantitative real-time RT-PCR (in German)
Simone Fleige & Michael W. Pfaffl (2007) Laborwelt, 2007 (5): 27-29, ISSN 1611–0854, (Editor: T. Gabrielczyk) Lehrstuhl für Physiologie, ZIEL, Technische Universität München, 85354 Freising Die mRNA Quantifizierung via real-time RT-PCR (qRT-PCR) findet heute Anwendung in einer Vielzahl der molekularbiologisch orientierten Labore. Die Methode birgt allerdings – gerade im Bereich der Prä-Analytik – zahlreiche Fehlerquellen. Vor allem die Messung der RNA Qualität führt bis dato noch ein Schattendasein. Das führt häufig zu ungenauen Ergebnissen oder zu erheblichen Variationen in den Expressionsergebnissen. Die Optimierung und Standardisierung der prä- und post-PCR stellen somit eine besondere Herausforderungen bei meiner validen mRNA Quantifizierung dar. Einmal mehr zeigt sich die Gesetzmäßigkeit, dass die Präzision der mRNA Genexpressionsanalyse durch die Quantität und Qualität des Ausgangsmaterials, der total RNA, signifikant beeinflusst wird. Die größte Verbesserung verspricht die Optimierung der Probenaufbereitung und die Bestimmung der RNA-Integrität, sowie die verbesserte Verrechung der erhaltenen real-time RT-PCR Daten. 
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