Microarray-based uncovering reference genes for quantitative real time PCR in grapevine under abiotic stress

被引：43

作者：

João L Coito

Margarida Rocheta

Luísa Carvalho

Sara Amâncio

机构：

[1] Centro de Botânica Aplicado À Agricultura, Departamento de Recursos Naturais, Universidade Técnica de Lisboa, Lisbon

来源：

BMC Research Notes | / 5卷 / 1期

关键词：

Grapevine; Microarray; Real time RT-PCR; Reference gene;

D O I：

10.1186/1756-0500-5-220

中图分类号：

学科分类号：

摘要：

Background: Quantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes. Results: Using the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in water stress, heat stress and the combination of both. Conclusions: Each method selected different gene combinations (PADCP + act, TIF + TIF-GTP and ubiq + act). However, as none of the combinations diverged significantly from the others used to normalize the expression of the putative stress marker genes, then any combination is suitable for data normalisation under the conditions tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray analysis. © 2012 Coito et al.; licensee BioMed Central Ltd.

引用

共 47 条

[1]

Bustin S.A., Benes V., Nolan T., Pfaffl M.W., Quantitative real-time RT-PCR - A perspective, Journal of Molecular Endocrinology, 34, 3, pp. 597-601, (2005)

[2]

Gachon C., Mingam A., Charrier B., Real-time PCR: What relevance to plant studies?, Journal of Experimental Botany, 55, 402, pp. 1445-1454, (2004)

[3]

Ginzinger D.G., Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream, Experimental Hematology, 30, 6, pp. 503-512, (2002)

[4]

Guenin S., Mauriat M., Pelloux J., Van Wuytswinkel O., Bellini C., Gutierrez L., Normalization of qRT-PCR data: The necessity of adopting a systematic, experimental conditions-specific, validation of references, J Exp Bot, 60, pp. 487-493, (2009)

[5]

Czechowski T., Stitt M., Altmann T., Udvardi M.K., Scheible W.-R., Genome-wide identification and testing of superior reference genes for transcript normalization in arabidopsis, Plant Physiology, 139, 1, pp. 5-17, (2005)

[6]

Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F., Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes, Genome Biol, 3, (2002)

[7]

Barsalobres-Cavallari C.F., Severino F.E., Maluf M.P., Maia I.G., Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions, BMC Mol Biol, 10, (2009)

[8]

Artico S., Nardeli S.M., Brilhante O., Grossi-De-Sa M.F., Alves-Ferreira M., Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data, BMC Plant Biol, 10, (2010)

[9]

Derveaux S., Vandesompele J., Hellemans J., How to do successful gene expression analysis using real-time PCR, Methods, 50, pp. 227-230, (2010)

[10]

Bustin S.A., Beaulieu J.-F., Huggett J., Jaggi R., Kibenge F.S.B., Olsvik P.A., Penning L.C., Toegel S., MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments, BMC Mol Biol, 11, (2010)

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