Abstract
In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.
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Abbreviations
- GAPDH:
-
Glyceraldehyde-3-phosphate-dehydrogenase
- eIF4AII:
-
Eukaryotic translation initiation factor 4AII
- 18S rRNA:
-
18S ribosomal RNA
- LEA:
-
Late Embryogenesis Abundant
- ABA:
-
Abscisic acid
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Acknowledgements
This research was supported by Fundação para a Ciência e a Tecnologia (FCT) and the III Framework Program of the EC through grants SFRH/BD/3135/2000 (SG) and SFRH/BPD/17902/2004 (CM) and through project POCTI/AGR/46283/2002. We thank Dr Concepción Ávila (Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias-Instituto Andaluz de Biotecnología, Universidad de Málaga, Spain), for providing primer sequence information regarding eIF-4AII gene. Estação Florestal Nacional (EFN) is acknowledged for making plant material available. JC acknowledges support from the National Science Foundation Plant Genome Program, Award No. 0217594.
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Gonçalves, S., Cairney, J., Maroco, J. et al. Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis. Planta 222, 556–563 (2005). https://doi.org/10.1007/s00425-005-1562-0
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DOI: https://doi.org/10.1007/s00425-005-1562-0