Molecular Breeding

, Volume 23, Issue 4, pp 607–616

Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR

  • Fernanda Cruz
  • Samara Kalaoun
  • Paula Nobile
  • Carlos Colombo
  • Juliana Almeida
  • Leila M. G. Barros
  • Eduardo Romano
  • Maria Fátima Grossi-de-Sá
  • Maité Vaslin
  • Marcio Alves-Ferreira
Article

DOI: 10.1007/s11032-009-9259-x

Cite this article as:
Cruz, F., Kalaoun, S., Nobile, P. et al. Mol Breeding (2009) 23: 607. doi:10.1007/s11032-009-9259-x

Abstract

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus drought-stressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here.

Keywords

Reference genes Coffee Drought stress Development Gene expression Real-time PCR qPCR 

Supplementary material

11032_2009_9259_MOESM1_ESM.doc (42 kb)
Supplemental Table 1 Primer sequences and efficiencies estimated by the Miner software. Independent efficiencies ± standard deviation (SD) were calculated for every different experimental situation evaluated (DOC 42 kb)

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Fernanda Cruz
    • 1
  • Samara Kalaoun
    • 1
  • Paula Nobile
    • 2
  • Carlos Colombo
    • 2
  • Juliana Almeida
    • 3
  • Leila M. G. Barros
    • 3
  • Eduardo Romano
    • 3
  • Maria Fátima Grossi-de-Sá
    • 3
  • Maité Vaslin
    • 4
  • Marcio Alves-Ferreira
    • 1
  1. 1.Department of Genetics, Center of Health SciencesFederal University of Rio de Janeiro (UFRJ)Rio de JaneiroBrazil
  2. 2.Center of GeneticsAgronomic Institute of CampinasCampinasBrazil
  3. 3.EMBRAPA Recursos Genéticos e BiotecnologiaBrasíliaBrazil
  4. 4.Microbioloy InstituteFederal University of Rio de Janeiro (UFRJ)Rio de JaneiroBrazil