Abstract
Quantitative real-time PCR (RT-qPCR) techniques have revolutionized gene expression analyses. To obtain accurate results, raw RT-qPCR results need to be normalized by using endogenous reference genes whose expression is assumed invariable in all studied samples. However, there are no universal reference genes, and candidate genes need to be evaluated for each experimental condition. In this work, we tested a set of possible reference genes for use in different organs and tissues of Pinus pinaster (needles from adult trees and different organs and developmental stages of seedlings). The putative reference genes were selected using microarray analyses and from those commonly used in previous works. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. To highlight the importance of these rules, 10 alternative primer pairs to be evaluated in pine samples were designed by following or not following the MIQE guidelines. Twenty-four candidate reference genes were tested in pine needles and 14 were also tested in pine seedlings. In both cases, valid reference genes were found, but differences in the stability and expression levels were also observed. Furthermore, a few of the best genes had unknown functions. The five most stable genes in the pine seedlings as well as four new candidate reference genes were evaluated in isolated tissues using laser capture microdissection. The results showed that the appropriate reference genes in different maritime pine organs were not invariable when sourced from the different tissues forming the organs.
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Acknowledgments
This work was supported by the European Commission Seventh Framework grant PROCOGEN (FP7-KBBE-2011-5), the Ministerio de Economía y Competitividad grant (BIO2015-69285-R), and Junta de Andalucía (BIO2012-0474).
Data archiving statement
Primer sequences are provided in Tables 1 and 2. Transcripts sequences are deposited in SustainPineDB (http://www.scbi.uma.es/sustainpinedb/home_page). Microarray data are available in NCBI GEO database (GSE55868).
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Communicated by P. Ingvarsson
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ESM 1
Supplemental Data S1. Cq values for the primer efficiency calculation. Four different calculations were included (PDF 124 kb)
ESM 2
Supplemental Data S2. Cq values for MIQE and Non-MIQE compliant primers in the all adult needle samples. The table includes the stability index calculated in geNorm (PDF 209 kb)
ESM 3
Supplemental Data S3. Cq values for MIQE compliant primers in the adult needle samples. The table includes the stability index calculated in geNorm, NormFinder, and BestKeeper (PDF 189 kb)
ESM 4
Supplemental Data S4. Cq values for MIQE compliant primers in the embryo and seedling samples. The table includes the stability index calculated in geNorm, Normfinder and Bestkeeper. (PDF 190 kb)
ESM 5
Supplemental Data S5. Cq values for the three better candidate reference genes in different physiological conditions and tissues. The table includes the stability index calculated in geNorm, NormFinder, and BestKeeper (PDF 208 kb)
ESM 6
Supplemental Data S6. Cq values for MIQE compliant primers at Laser Capture Microdissection samples. The table includes the stability index calculated in geNorm, NormFinder, and BestKeeper (PDF 192 kb)
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Granados, J.M., Ávila, C., Cánovas, F.M. et al. Selection and testing of reference genes for accurate RT-qPCR in adult needles and seedlings of maritime pine. Tree Genetics & Genomes 12, 60 (2016). https://doi.org/10.1007/s11295-016-1018-7
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DOI: https://doi.org/10.1007/s11295-016-1018-7