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OVERVIEW

REVIEW:  RNA integrity and the effect on the real-time qRT-PCR performance.

Molecular Aspects of Medicine 27 (2006) 126–139
Simone Fleige a, 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.


Newly added RNA intergrity publication:
Talks:
WEBINAR - Audio slide shows

DEGRADOMETER Software

Degradometer Manual

Download Degradometer software version 1.41.zip

PAPER:  Chipping away at the chip bias: RNA degradation in microarray analysis.

POSTER:  Estimation and Reduction of the Bias Caused by RNA Degradation in Microarray Analysis.
Measurements of gene expression are based on the assumption that the analyzed RNA sample closely resembles the amount of transcripts in vivo. Established knowledge that transcripts of different genes possess different stabilities suggests that degradation of RNA occurring during the isolation procedure is also non-uniformly distributed among different RNA species. Indeed, comparison of RNA samples of different degrees of degradation shows that up to 75% of microarray-based measurements of differential gene expression can be caused by degradation bias alone1. We demonstrate that analysis of capillary-electrophoresis data does allow reproducible characterization of RNA degradation and its differentiation from apoptosis-associated RNA cleavage. Degradometer software for quantification of RNA integrity is available on our website (www.dnaarrays.org). Our results suggest that comparison of RNA samples of similar integrity eliminates skewed results of differential gene expression. Consequently, information about quantification of RNA integrity will help to improve reproducibility of
microarray results.

The External RNA Control Consortium (ERCC) is an ad-hoc group with approximately 70 members from private, public, and academic organizations. The group was initiated in 2003 to develop a set of external RNA control transcripts that can be used to assess technical performance in gene expression assays. The external RNA controls will be added after RNA isolation, but prior to cDNA synthesis. They are being designed to evaluate whether the results for a given experiment are consistent with defined performance criteria. All ERCC work is intended to apply to quantitative, real-time reverse transcriptase polymerase chain reaction (QRT-PCR) assays as well as one-color and two-color microarray experiments.

The ERCC has worked together to define the desired properties of the transcripts, general protocols for their application, and an analysis scheme for performance assessment. In December 2003, the group developed a specification document that was discussed and refined in a public workshop at the National Institute of Standards and Technology (NIST). Protocols for the use of external RNA controls in clinical applications are included in the Molecular Methods 16-P document from the Clinical and Laboratory Standards Institute, and were developed in a formal, accredited, open, consensus forum including several ERCC members. The analysis approach was developed in a public workshop at NIST in June 2004, and is based upon the measurement of pooled transcripts at known concentrations.

In the past year, the ERCC has refined specifications, generated and collected control sequences, evaluated optimal polyadenylated (polyA) tail length and identified a path forward for access and distribution of the controls, as descibed in "Proposed Methods for Testing and Selecting the ERCC External RNA Controls". Input on the proposed methods is being solicited by e-mail at ercc@nist.gov

 
RNA Integrity number Database - RINdb

http://www.chem.agilent.com/rin/_rinSearch.aspx

The RNA Integrity database (RINdb) is a freely accessible collection of records containing electropherograms, RIN and other sample metadata. Scientists can query the database to prescreen methods for a new experiment, compare RIN thresholds and validate own results. All users can contribute records of their own.

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