Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1α for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1α and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis.

Department of Molecular Biology Crop Research Institute v v i Drnovská 507 Ruzyne 161 06 Prague 6 Czech Republic

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Curr Opin Biotechnol. 2005 Apr;16(2):123-32 PubMed

PLoS One. 2011 May 12;6(5):e19434 PubMed

Mol Med Today. 1998 Sep;4(9):389-96 PubMed

Plant J. 2004 Apr;38(2):366-79 PubMed

BMC Plant Biol. 2008 Nov 07;8:112 PubMed

Mol Genet Genomics. 2012 Jan;287(1):1-19 PubMed

BMC Plant Biol. 2009 Jul 02;9:84 PubMed

Funct Integr Genomics. 2011 Jun;11(2):307-25 PubMed

Cancer Res. 2004 Aug 1;64(15):5245-50 PubMed

Genome Biol. 2002 Jun 18;3(7):RESEARCH0034 PubMed

Nucleic Acids Res. 2007 Jul;35(Web Server issue):W71-4 PubMed

BMC Mol Biol. 2008 May 19;9:49 PubMed

Animal. 2010 Jun;4(6):853-60 PubMed

J Exp Bot. 2011 Aug;62(13):4675-89 PubMed

New Phytol. 2009;182(1):213-228 PubMed

Mol Genet Genomics. 2012 Feb;287(2):167-76 PubMed

Mech Ageing Dev. 2010 Feb;131(2):89-95 PubMed

Plant Mol Biol. 2007 Mar;63(5):679-88 PubMed

J Exp Bot. 2005 Nov;56(421):2907-14 PubMed

Biotechniques. 2005 Jul;39(1):75-85 PubMed

PLoS One. 2012;7(7):e41886 PubMed

J Food Prot. 2010 Apr;73(4):688-94 PubMed

J Biochem Biophys Methods. 2000 Nov 20;46(1-2):69-81 PubMed

Biotechnol Lett. 2004 Mar;26(6):509-15 PubMed

Plant Physiol. 2004 Jan;134(1):224-36 PubMed

Plant Cell. 2008 Aug;20(8):2117-29 PubMed

Plant Cell Rep. 2011 Apr;30(4):641-53 PubMed

The up-regulation of elongation factors in the barley leaf and the down-regulation of nucleosome assembly genes in the crown are both associated with the expression of frost tolerance