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Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions

Marine Biology volume 159, pages 1269–1282 (2012)Cite this article

Abstract

The stability on the expression level of putative reference genes (RGs) for reverse transcriptase-quantitative PCR (RT-qPCR) was tested in the seagrass Posidonia oceanica for plants collected in different environmental conditions (at different depths and in different salinity and pH). Besides adult plants, seedlings were also used for pH and salinity experiments. The putative RGs encompassed the most frequently used in other species to normalize RT-qPCR in a range of experimental conditions. Assignment of the best RGs has been performed with three Excel-based applets, BestKeeper, geNorm and NormFinder, and the results showed that best RGs may change depending on the experimental conditions tested, plant age and on the software utilized. Preferred sets of RGs are proposed for each of the conditions and a general procedure concerning their selection is discussed. In addition, expression levels of four genes involved in plant response to light, salinity and pH variation have also been evaluated.

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Acknowledgments

The authors thank the staff of the Molecular Biology Service of Stazione Zoologica Anton Dohrn for their technical support in laboratory experiments, M. Lorenti and V. Rando for their help in collecting environmental data, M.C. Buia for providing support in the Lacco Ameno sampling site and the anonymous reviewers for their constructive suggestions.

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Authors and Affiliations

  1. Università Della Calabria, Via P. Bucci, Arcavacata di Rende, Cosenza, Italy

    Ilia Anna Serra, Silvia Nicastro & Anna Maria Innocenti

  2. Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy

    Chiara Lauritano, Emanuela Dattolo, Andrea Puoti & Gabriele Procaccini

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  1. Ilia Anna Serra
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  2. Chiara Lauritano
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  3. Emanuela Dattolo
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  4. Andrea Puoti
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  5. Silvia Nicastro
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  6. Anna Maria Innocenti
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  7. Gabriele Procaccini
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Corresponding author

Correspondence to Gabriele Procaccini.

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Communicated by T. Reusch.

Ilia Anna Serra, Chiara Lauritano share equal responsibility.

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227_2012_1907_MOESM1_ESM.pdf

Online resource Ranking of the best reference genes (RGs) obtained with BestKeeper, geNorm and NormFinder in the different experimental conditions: adults and young exposed to salinity, adults and young exposed to pH, adults exposed to salinity, adults exposed to pH, young exposed to pH, young exposed to salinity, adults exposed to different depths, young exposed to salinity and pH and adults plants exposed to different depths, salinity and pH. a) Results of the BestKeeper analysis on the six putative Reference Genes tested. Number of samples (n), geometric mean of Ct (GM), arithmetic mean of Ct (AM), extreme values of Ct (Min and Max), standard deviation of the Ct (SD), coefficient of variance, expressed as a percentage on the Ct level (CV), extreme values of expression levels, expressed as an absolute x-fold over- or under-regulation coefficient (Min [x-fold] and Max [x-fold]), and standard deviation of the absolute regulation coefficients (SD [± x-fold]), are given. b) The stepwise exclusion of genes that are more variable among samples using the geNorm program. More stable genes are indicated by the arrow; c) Pairwise variation (V) to evaluate the effect of adding another RG to the best couple already analyzed using geNorm (e.g., adding a third gene V2/3, a fourth V3/4, etc.). The inclusion of additional RGs was not required below the cutoff value of 0.15; d) The NormFinder algorithm ranks the candidate RGs according to their expression stability. Lower stability values indicate more stable genes (PDF 328 kb)

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Serra, I.A., Lauritano, C., Dattolo, E. et al. Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions. Mar Biol 159, 1269–1282 (2012). https://doi.org/10.1007/s00227-012-1907-8

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  • DOI: https://doi.org/10.1007/s00227-012-1907-8

Keywords

  • Reference Gene
  • Candidate Reference Gene
  • Salinity Variation
  • Subtractive Suppression Hybridization Library
  • geNorm Analysis