Abstract
Gene expression analysis by means of RT-qPCR is a highly sensitive technique. However, this requires an accurate protocol for the whole procedure from sampling to data analysis. We have optimized this protocol specifically for the analysis of plant tissues. Special attention is paid to RNA quality and integrity and to the appropriate setup of the assays in order to be compliant with the MIQE guidelines. This protocol was already successfully applied in ten different plant species.
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References
Luypaert G, Witters J, Van Huylenbroeck J et al (2017) Induced expression of selected plant defence related genes in pot azalea, Rhododendron simsii hybrid. Euphytica 213:227
Die JV, Roman B (2012) RNA quality assessment: a view from plant qPCR studies. J Exp Bot 63:6069–6077
De Keyser E, Desmet L, Van Bockstaele E et al (2013) How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression analysis in an azalea (Rhododendron simsii hybrids) mapping population. BMC Mol Biol 14:13. https://doi.org/10.1186/1471-2199-14-13
Guttierrez L, Mauriat M, Guénin S, Pelloux J et al (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription – polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6:609–618
Czechowski T, Stitt M, Altmann T et al (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17
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
Nolan T, hands RE, Ogunkolade BW et al (2006) SPUD: a qPCR assay for the detection of inhibitors in nucleic acid preparations. Anal Biochem 351:308–310
Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034.1–research 0034.11
Hellemans J, Mortier G, De Paepe A et al (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19
Ruijter JM, Ramakers C, Hoogaars WMH et al (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37:e45–e45
Berruti A, Christiaens A, De Keyser E et al (2015) Cold treatment breaks dormancy but jeopardizes flower quality in Camellia japonica L. Front Plant Sci 6:983
Ouyang L, Leus L, De Keyser E et al (2019) Seasonal changes in cold hardiness and carbohydrate metabolism in four garden rose cultivars. J Plant Physiol 232:188–199
Merlaen B, De Keyser E, Van Labeke MC (2018) Identification and substrate prediction of new Fragaria x ananassa aquaporins and expression in different tissues and during strawberry fruit development. Hortic Res 5:20
Dierck R, Leus L, Dhooghe E et al (2018) Branching gene expression during chrysanthemum axillary bud outgrowth regulated by strigolactone and auxin transport. Plant Growth Regul 86:23–36
Luu-The V, Paquet N, Calvo E, Cumps J (2005) Improved realt-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction. BioTechniques 38:287–293
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De Keyser, E., Desmet, L., Losschaert, M., De Riek, J. (2020). A General Protocol for Accurate Gene Expression Analysis in Plants. In: Biassoni, R., Raso, A. (eds) Quantitative Real-Time PCR. Methods in Molecular Biology, vol 2065. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9833-3_9
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DOI: https://doi.org/10.1007/978-1-4939-9833-3_9
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9832-6
Online ISBN: 978-1-4939-9833-3
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