NDLI: Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Content Provider	Springer Nature Link
Author	Fleige, Simone Walf, Vanessa Huch, Silvia Prgomet, Christian Sehm, Julia Pfaffl, Michael W.
Copyright Year	2006
Abstract	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.
Starting Page	1601
Ending Page	1613
Page Count	13
File Format	PDF
ISSN	01415492
Journal	Biotechnology Letters
Volume Number	28
Issue Number	19
e-ISSN	15736776
Language	English
Publisher	Kluwer Academic Publishers
Publisher Date	2006-08-10
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Gene expression study Ct mRNA qRT-PCR real-time RT-PCR RNA integrity RNA integrity number (RIN)
Content Type	Text
Resource Type	Article
Subject	Medicine Bioengineering Applied Microbiology and Biotechnology Biotechnology

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Original paper comparison of relative mrna quantification models and the impact of rna integrity in quantitative real-time rt-pcr.

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

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