Abstract
RNA quality control is a crucial step in guaranteeing integer nondegraded RNA and receiving meaningful results in gene expression profiling experiments, using micro-array, RT-qPCR (Reverse-Transcription quantitative PCR), or Next-Generation-Sequencing by RNA-Seq or small-RNA Seq. Therefore, assessment of RNA integrity and purity is very essential prior to gene expression analysis of sample RNA to ensure the accuracy of any downstream applications. RNA samples should be nondegraded or fragmented and free of protein, genomic DNA, nucleases, and enzymatic inhibitors. Herein we describe the current state-of-the-art RNA quality assessment by combining UV/Vis spectrophotometry and microfluidic capillary electrophoresis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Becker C, Hammerle-Fickinger A, Riedmaier I et al (2010) mRNA and microRNA quality control for RT-qPCR analysis. Methods 50:237–243. doi:10.1016/j.ymeth.2010.01.010
Fleige S, Pfaffl MW (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 27:126–139
Vermeulen J, Prete K, de Lefever S (2011) Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res 39(9):e63. doi:10.1093/nar/gkr065
Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582
Pérez-Novo CA, Claeys C, Speleman F et al (2005) Impact of RNA quality on reference gene expression stability. BioTechniques 39:54–56
Holland NT, Smith MT, Eskenazi B et al (2003) Biological sample collection and processing for molecular epidemiological studies. Mutat Res 543:217–234
Schoor O, Weinschenk T, Hennenlotter J et al (2003) Moderate degradation does not preclude microarray analysis of small amounts of RNA. BioTechniques 35(1192–1196):1198–1201
Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Pfaffl MW (2005) Nucleic acids: mRNA identification and quantification. In: Worsfold P, Townshend A, Poole CF (eds) Encyclopedia of analytical science, 2nd edn. Elsevier Academic Press, Amsterdam, Boston, pp 417–426
Manchester KL (1996) Use of UV methods for measurement of protein and nucleic acid concentrations. BioTechniques 20:968–970
Nolan T, Hands RE, Ogunkolade W et al (2006) SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Anal Biochem 351:308–310
Fleige S, Walf V, Huch S et al (2006) Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett 28:1601–1613
Mueller O, Lightfoot S, Schroeder A et al (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3. doi:10.1186/1471-2199-7-3
Miller CL, Diglisic S, Leister F et al (2004) Evaluating RNA status for RT-PCR in extracts of postmortem human brain tissue. BioTechniques 36:628–633
Santiago TC, Purvis IJ, Bettany AJ et al (1986) The relationship between mRNA stability and length in Saccharomyces cerevisiae. Nucleic Acids Res 14:8347–8360
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kirchner, B., Paul, V., Riedmaier, I., Pfaffl, M.W. (2014). mRNA and microRNA Purity and Integrity: The Key to Success in Expression Profiling. In: Biassoni, R., Raso, A. (eds) Quantitative Real-Time PCR. Methods in Molecular Biology, vol 1160. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0733-5_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0733-5_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0732-8
Online ISBN: 978-1-4939-0733-5
eBook Packages: Springer Protocols