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Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions


Casuarina glauca is a model actinorhizal plant species that establishes N2-fixing symbiosis with Frankia bacteria. This plant is highly resilient to extreme environments, being commonly found in saline zones. Gene expression studies by quantitative real-time polymerase chain reaction (qRT-PCR) constitute a powerful tool to analyze the mechanisms underlying plant stress-tolerance. One of the crucial steps of this technique is the selection and validation of reference genes to produce accurate data. In this work we report on the evaluation of a set of ten reference genes to be used in qRT-PCR studies in C. glauca grown under high salt concentrations, following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Five independent methods (geNorm, NormFinder, BestKeeper, Coefficient of Variance, and ReFinder) were used to evaluate gene stability. According to the results, the calibration of qRT-PCR reactions with the most versus the least stable reference genes produced different expression patterns of C. glauca stress responsive genes (CgCS and CgAPX). The same was observed when data was normalized with one, two or three stable reference genes. These study constitutes a baseline for accurate qRT-PCR analysis in C. glauca exposed to high salt concentrations which should include the use of at least two stable reference genes.

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This work was supported by Fundação para a Ciência e Tecnologia under the scope of the project PTDC/AGR-FOR/4218/2012 and grant SFRH/BPD/78619/2011 (P. Batista-Santos). The authors acknowledge Paula Alves for lab assistance.

Competing interests

The authors declare that they have no competing interests.

Author’s contributions

MdC performed the experimental work and produced the manuscript draft; ND contributed to the experimental work and to the manuscript draft; PB-S contributed to the experimental work; JCR contributed to the study design; AR-B designed and coordinated the experimental work and was involved in drafting the manuscript. MdC, AR-B and JCR gave thorough review and final approval of the manuscript.

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Correspondence to Ana I. Ribeiro-Barros.

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Key message

Folowing the MIQE guidelines, we have defined the most appropriate number and classes of reference genes for qRT-PCR studies in symbiotic and non-symbiotic C. glauca exposed to high salinity conditions.

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Online Resource 1
figure 9

Experimental outline. (GIF 387 kb)

Online Resource 3
figure 10

Melting Curve from qRT-PCR of each Reference Genes. a) Ap47; b) GAPDH; c) Ef-1; d) Elf-4a; e) S24; f) Tubulin; g) Apt; h) Ubiquitin Checklist for MIQE guidelines. (GIF 526 kb)

Online Resource 6
figure 11

Diagram that summarizes the top two genes for qRT-PCR in each tissue analyzed. The roots and branchlets were collected from NOD− plants, and nodules and branchlets material from symbiotic (NOD+) plants. (GIF 60 kb)

High resolution image (TIFF 2106 kb)

Online Resource 2

Samples used for total assay and intra-group analyses (DOCX 16 kb)

High resolution image (TIFF 1274 kb)

Online Resource 4

qPCR products sequencing results (DOCX 13 kb)

Online Resource 5

Checklist for MIQE guidelines. (XLSX 16 kb)

High resolution image (TIFF 100 kb)

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da Costa, M., Duro, N., Batista-Santos, P. et al. Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions. Symbiosis 66, 21–35 (2015).

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  • Actinorhizal nodules
  • Casuarina glauca
  • Frankia
  • qRT-PCR
  • Reference
  • Salt stress