The next generation in RNA QC is here - QIAxcel Pure Excellence
Analyze RNA quality with confidence and convenience
RIS indicates RNA integrity. Streamlined RNA analysis using the QIAxcel Advanced System.

QIAxcel Advanced and new QIAxcel ScreenGel Software provide:
  • Objective RNA quality measurement with the new RNA Integrity Score (RIS)
  • Rapid analysis of up to 96 samples without manual intervention
  • Safety, convenience, and time-savings with ready-to-use gel cartridges
  • User-friendly operation with no tedious chip loading procedure
  • Process profiles to standardize applications and reduce handling errors

QIAxcel Advanced fully automates sensitive, high-resolution capillary electrophoresis of up to 96 samples per run, streamlining your workflow and reducing time to result. The new RNA Integrity Score (RIS) is an objective quality measurement for eukaryotic RNA samples, which enables fast and easy quality control of RNA integrity. QIAxcel takes ease of use to a new level — just load the gel cartridge and buffer tray, place your samples into the instrument, select the process profile, and go. Say good-bye to tedious slab gel preparation and chip loading procedures, and say hello to QIAxcel Advanced!



For effortless DNA, RNA, and protein analysis:
  • Rapid analysis of up to 96 samples without manual intervention
  • Safety and convenience with ready-to-use gel cartridges
  • Accurate analysis of low concentration nucleic acids with 3–5 bp resolution
  • User-friendly analysis software that supports electronic signatures and records
  • Objective RNA quality measurement with new RNA Integrity Score (RIS)
 
The revolutionary QIAxcel Advanced system replaces traditional, labor-intensive gel analysis of DNA, RNA, and proteins - streamlining workflows and reducing time to result. The QIAxcel Advanced system fully automates sensitive, high-resolution capillary electrophoresis of up to 96 samples per run. DNA fragment analysis of 12 samples can be performed in as little as 3 minutes. Ready-to-run gel cartridges allow 96 samples to be analyzed with a minimum of hands-on interaction, reducing manual handling errors and eliminating the need for tedious gel preparation. User-friendly QIAxcel ScreenGel software ensures convenient analysis and documentation of data. The QIAGEN RNA Integrity Score (RIS) provides an objective quality measurement of the analyzed samples and allows easy interpretation of sample integrity.






Download the brochure and find out how you can achieve effortless DNA, RNA, and protein analysis with QIAxcel Advanced and QIAxcel ScreenGel Software.



QIAxpert System

For accelerated DNA, RNA, and protein quantification and quality control
  • Quantify nucleic acids from up to 16 samples in less than 2 minutes
  • Discriminate between molecules of interest using unique spectral protocols
  • Determine specific amounts of DNA, RNA, and other contaminating fractions
  • Rapidly perform analyses via the intuitive, full-color integrated touchscreen
  • Generate comprehensive reports to view on any computer or smart device
QIAxpert is an innovative high-speed microfluidic UV/VIS spectrophotometer which profiles sample content to differentiate between DNA and RNA and sample impurities. QIAxpert is fast and easy to use, analyzing up to 16 samples within 2 minutes -  just pipet samples onto the QIAxpert slide, place it into the reader, and select the pre-programmed method on the integrated touchscreen. QIAxpert is the right tool for any fast-paced lab performing nucleic acid quantification and quality control.





Bioanalyzer 2100  &  Experion

Microfluidics
deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-milimeter, scale. It is a multidisciplinary field intersecting engineering, physics, chemistry, microtechnology and biotechnology, with practical applications to the design of systems in which such small volumes of fluids will be used. Microfluidics has emerged in the beginning of the 1980s and is used in the development of inkjet printheads, DNA chips, lab-on-a-chip technology, micro-propulsion, and micro-thermal technologies.

More info  =>  Company Bulletins  --  Ambion, Agilent Technologies, Bio-Rad, Qiagen
More info on our Lab on Chip sub-domain  =>  Lab-on-chip  sub-domain

RNA quality control in miRNA expression analysis
Christiane Becker, Martina Reiter, Michael W. Pfaffl
Agilent Technologies Application Note 5990-5557EN

It is generally known that total RNA quality has a distinct influence on the validity and reliability of quantitative PCR results. In addition, the recently published MIQE guidelines focus on the pre-PCR steps and state the importance of RNA quality assessment. Various studies showed the impairing effect of ongoing RNA degradation on mRNA expression results. Therefore, the verification of RNA integrity prior to downstream applications like RT-qPCR and mircroarrays is indispensable. A fast and reliable assessment of RNA integrity can be done with the Eukaryote Total RNA Nano Assay of the Agilent 2100 Bioanalyzer. The importance of RNA quality should also be considered in new applications such as the investigation of miRNA expression profiles. With the Agilent Small RNA Assay, Agilent is offering one of the few possibilities for selectively estimating miRNA before expression analysis. However, by now little is known about factors affecting miRNA analysis. Herein, the important impact of total RNA quality on quantification of mRNA and miRNA should be considered.

Comply with MIQE guidelines for qPCR  -  Agilent 2100 Bioanalyzer assessment of RNA integrity
by Ruediger Salowsky
Agilent Product Manager Bioanalyzer - RNA/DNA Solutions
Access Agilent eNewsletter, January 2010

Quantitative real-time polymerase chain reaction (qPCR) and microarray analysis have become essential for elucidating variations in gene expression. While guidelines that define the minimum information required for interpretation of microarray data have been available since 2001,[1] similar specifications for qPCR experiments have been developed only recently. In early 2009, a consortium of leading scientists who use qPCR, established specifications for the minimum information that you must report for a qPCR experiment that you wish to publish. These are the MIQE guidelines (for minimum information for publication of quantitative real-time PCR experiments). This article describes how the Agilent 2100 Bioanalyzer helps you meet these requirements.

Validation of  Lab-on-Chip  capillary electrophoresis systems for total RNA quality and quantity control
M.W. Pfaffl, S. Fleige & I. Riedmaier
Technical University of Munich, center of life and Food Sciences (Ziel), Physiology Weihenstephan, Freising, Germany
Biotechnol. & Biotechnol. 2008 22(3)  829-834
Download Paper

Purity and good RNA quality are important elements for the overall success of RNA based analysis methods like microarrays and real time qRT-PCR. There are two commercially available automated systems – the Experion (Bio-Rad Laboratories) and the 2100 Bioanalyzer (Agilent Technologies) – that provide both RNA sample quality and quantity analysis. In this study different aspects like the reproducibility and sensitivity of both systems were analyzed by determining the total RNA quality and quantity extracted from various bovine tissues. Regarding quantitation, the Experion is more sensitive than the 2100 Bioanalyzer. Both systems overstate the concentration by 19-29% compared to the photometric values. For RNA quality determination, both systems show highly comparable reproducibility. With the RNA integrity number (RIN) the 2100 Bioanalyzer offers an additional opportunity to quantify the RNA quality.

The focus on sample quality -- Influence of colon tissue collection on reliability of qPCR data.
Korenkova V, Slyskova J, Novosadova V, Pizzamiglio S, Langerova L, Bjorkman J, Vycital O, Liska V, Levy M, Veskrna K, Vodicka P, Vodickova L, Kubista M, Verderio P
Download -- Sci Rep. 2016 6: 29023

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.
Development and Validation of RQ -- An RNA Quality Indicator for the Experion Automated Electrophoresis System
by Bio-Rad -- Bulletin 5761





The AdvanCE™ FS Nucleic Acids Analyzer

The AdvanCE™ FS Nucleic Acids Analyzer is a fluorescence-based capillary electrophoresis instrument for both sizing and quantifying nucleic acids (DNA and RNA). By using a sensitive intercalating dye coupled with a powerful LED light source, AdvanCE™ FS Nucleic Acids Analyzer obviates the need for fluorescent labeled primers and can be used to separate dsDNA fragments and RNA (mRNA and total RNA). The AdvanCE™ FS Nucleic Acids Analyzer is the most flexible system on the market today, with the greatest sensitivity, the highest separation resolution over a wide range, the widest dynamic range and the fastest separation times.

This instrument comes available for customers who wish to run either 12 samples or 96 samples at a time.

Currently kits are available to:
Gels formulated to provide high resolution separation are also available for general fragment analysis, such as SSR/microsatellites, small and large PCR fragments (amplicons) and resolve fragments sized between 10 bp and 40,000 bp with resolution down to 2bp.

Assessment of RNA quality and RNA quantitation is critical for many molecular biology techniques. Manual assessment methods for both quantity and quality can use large amounts of precious materials and take a considerable amount of time to complete.

Several analytical instruments exist for the evaluation of RNA. However, these instruments either lack acceptable detection sensitivity or require significant hands-on time to complete chip loading and preparation. The AdvanCE FS process for RNA – either for total RNA and/or messenger RNA – is completely automated, requiring minimal hands on time.





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.

Our RNA integrity basis paper has been frequently cited by other researchers:  => 808 times until April 2016

mRNA and microRNA quality control for RT-qPCR analysis
Becker C, Hammerle-Fickinger A, Riedmaier I, Pfaffl MW.
Physiology-Weihenstephan, Technical University Munich, Freising, Germany.
Methods. 2010 50(4): 237-243

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency. RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity.

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR.
Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW.
Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL), Technical University Munich, Germany.
Biotechnol Lett. 2006 28(19): 1601-1613

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.


Our second RNA integrity basis paper has been frequently cited by other researchers:  => 386 times until April 2016

    http://Scholar.Google.com/



Measurable impact of RNA quality on gene expression results from quantitative PCR.
Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S, Hellemans J, Speleman F, Vandesompele J.
Nucleic Acids Res. 2011 39(9): e63

Compromised RNA quality is suggested to lead to unreliable results in gene expression studies. Therefore, assessment of RNA integrity and purity is deemed essential prior to including samples in the analytical pipeline. This may be of particular importance when diagnostic, prognostic or therapeutic conclusions depend on such analyses. In this study, the comparative value of six RNA quality parameters was determined using a large panel of 740 primary tumour samples for which real-time quantitative PCR gene expression results were available. The tested parameters comprise of microfluidic capillary electrophoresis based 18S/28S rRNA ratio and RNA Quality Index value, HPRT1 5'-3' difference in quantification cycle (Cq) and HPRT1 3' Cq value based on a 5'/3' ratio mRNA integrity assay, the Cq value of expressed Alu repeat sequences and a normalization factor based on the mean expression level of four reference genes. Upon establishment of an innovative analytical framework to assess impact of RNA quality, we observed a measurable impact of RNA quality on the variation of the reference genes, on the significance of differential expression of prognostic marker genes between two cancer patient risk groups, and on risk classification performance using a multigene signature. This study forms the basis for further rational assessment of reverse transcription quantitative PCR based results in relation to RNA quality.




RNA degradation compromises the reliability of microRNA expression profiling
David Ibberson, Vladimir Benes, Martina U Muckenthaler and Mirco Castoldi
Genomics Core Facility, EMBL, Meyerhofstraße 1 D-69117 Heidelberg, Germany;  Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 156, D-69120, Heidelberg, Germany;  Molecular Medicine Partnership Unit, Im Neuenheimer Feld 156, D-69120, Heidelberg, Germany
BMC Biotechnology 2009

MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles. Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP) or microRNA-specific real-time quantitative PCR (miQPCR). Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays. MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven.

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.

Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses.
Hammerle-Fickinger A, Riedmaier I, Becker C, Meyer HH, Pfaffl MW, Ulbrich SE.
Biotechnol Lett. 2010 32(1): 35-44   
Supplement - Biotechnol Lett. 2010 32(1): 35-44
Physiology Weihenstephan, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354, Freising, Germany.

The benefit and precision of blood diagnosis by quantitative real-time PCR (qPCR) is limited by sampling procedures and RNA extraction methods. We have compared five different RNA extraction protocols from bovine blood regarding RNA and miRNA yield, quality, and most reproducible data in the qRT-PCR with the lowest point of quantification. Convincing results in terms of highest quantity, quality, and best performance for mRNA qPCR were obtained by leukocyte extraction following blood lysis as well as extraction of PAXgene stabilized blood. The best microRNA qPCR results were obtained for samples extracted by the leukocyte extraction method.

Einfluss der RNA Integrität 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.

Comparison of two available platforms for determination of RNA quality
I. Riedmaier, M. Bergmaier and M.W. Pfaffl
Technische Universitat Munchen, Physiology Weihenstephan, Freising, Germany
Biotechnol. & Biotechnol. Eq. 2010, 24(4): 2154-2159

The integrity of RNA is a very critical aspect regarding downstream RNA based quantitative analysis like RT-qPCR. Low-quality RNA can compromise the results of such experiments. Today automated lab-on-chip capillary electrophoresis allows rapid RNA quality and quantity determination, e.g. 2100 Bioanalyzer (Agilent Technologies) and the Experion (Bio-Rad). Both platforms determine RNA quality using a numerical system which represents the integrity of RNA. The Bioanalyzer offers the RIN algorithm (RNA Integrity Number) on the Bioanalyzer 2100 and Bio-Rad developed a new Experion software version that offers an algorithm for calculating the RNA Quality Index (RQI).The aim of this study was to compare both systems regarding sensitivity, reproducibility, linearity and the influence of individual tissue extractions and different chip runs on RNA quality and quantity determination.Overall it was confirmed that both algorithms are very comparable and beneficial for the determination of RNA quality for downstream applications. The Experion showed slightly better results regarding reproducibility and absolute sensitivity, whereas the 2100 Bioanalyzer showed a higher linearity.

MIQE Guidelines
RNA Qualitätskontrolle in der Genexpressionsanalytik   (in German)
Christiane Becker, Irmgard Riedmaier, Michael W. Pfaffl
BIOspektrum - Special RNA Technologien 2009 (5): 512 - 515

Abstrakt (D) - Die Qualität des Probenmaterials, also der Gesamt-RNA, hat einen markanten Einfluss auf die Richtigkeit der quantitativen RT-PCR. Die Überprüfung der RNA Qualität vor einer Expressionsmessung ist unabdingbar, um verlässliche RT-qPCR Expressionsergebnisse zu erhalten.
Abstract (E) - The integrity of total RNA has a distinct influence on the accuracy of RT-qPCR. Quality assessment is an essential step for the evaluation of reliable results in gene expression analysis.


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