CSIRO PUBLISHING Functional Plant Biology,2012,39,342-350 http://dx.doi.org/10.1071/FP11246 Validation of reference genes for real-time quantitative PCR normalisation in non-heading Chinese cabbage Dong XiaoB,Ning-Wen ZhangB,Jian-Jun ZhaoB.C,Guusje BonnemaB.D and Xi-Lin HouD AState Key Laboratory of Crop Genetics and Germplasm Enhancement;Horticultural College, Nanjing Agricultural University,Nanjing,Jiangsu 210095,China. BLaboratory of Plant Breeding,Wageningen University,The Netherlands. CHorticultural College,Hebei Agricultural University,Baoding,Hebei,China. PCorresponding author.Emails:guusje.bonnema@wur.nl;hxl@njau.edu.cn Abstract.Non-heading Chinese cabbage is an important vegetable crop that includes pak choi,caixin and several Japanese vegetables like mizuna,mibuna and komatsuna.Gene expression studies are frequently used to unravel the genetics of complex traits and in such studies the proper selection of reference genes for normalisation is crucial.We assessed the expression of 13 candidate reference genes including ACTIN,ACTIN-1,ACTIN-2,GAPDH,Tub_a,CyP,EFI-a,18SrRNA, UBO,UBC30,PPR,PP24 and MDH.Their expression stabilities were analysed using two programs,geNorm and NormFinder,in 20 different samples that represent four strategic groups.Results showed that no single gene was uniformly expressed in all tested samples.ACTINand CyP are proposed as good reference genes when studying developmental stages. CyP,Tub_a and UBC30 are good reference genes when studying different tissues(from flowering to seed set).CyP and Tub_a are the most stable reference genes under biotic stress treatments using the fungi Peronospora parasitica and Alternaria brassicicola.UBC30,EF/-a and ACTIN are recommended for normalisation in abiotic stress studies,including hormone,salt,drought,cold and heath treatments.Moreover,at least five reference genes(ACTIN,CyP,UBC30,EFI-a and UBO)are required for accurate qRT-PCR data normalisation when studying gene expression across all tested samples. Additional keywords:Brassica rapa ssp.chinensis,gene expression,qRT-PCR,reference genes. Received 29 October 2011,accepted 7 March 2012,published online 24 April 2012 Introduction expression level under all the experimental situations tested The quantification of mRNA (mRNA)transcript levels has (Kim et al.2003;Ding et al.2004;Argyropoulos et al.2006). become an important research tool in recent years.Changes in Use of inappropriate reference genes in relative quantification mRNA transcript levels are crucial during plant developmental of gene expression profiles may lead to erroneous normalisation processes,between different tissues and under changing and consequently,misinterpretation of the results.Therefore,it environmental conditions.Real-time quantitative PCR (qRT- is essential to validate the expression stability of reference genes PCR)has become the most popular method to quantify mRNA in each experimental system transcription levels and to validate whole-genome microarray In plant research, glyceraldehyde-3-phosphate data because of its outstanding accuracy,broad dynamic range dehydrogenase (GAPDH),B-ACTIN (ACTIN),tubulin a and high sensitivity not only in the fields of molecular medicine, (Tub_a)and 18S rRNA were considered to have a constant biotechnology,microbiology and molecular diagnostics but also expression level and as a consequence have been widely used in plant research (Vandesompele et al.2002;Jian et al.2008; as reference genes for normalisation of gRT-PCR data in Paolacci et al.2009).Estimating the expression levels of target various experimental conditions (Kim et al.2003;Ding et al. genes of interest by qRT-PCR depends on endogenous control 2004;Jian et al.2008;Lovdal and Lillo 2009).However,it has genes to normalise qRT-PCR;control genes are also called also been reported that the transcript levels of these genes can reference genes or housekeeping genes (HKGs)(Wierschke change significantly under different experimental conditions et al.2010;Martinez-Beamonte et al.2011).HKGs play a (Czechowski et al.2005;Terrier and Glissant et al.2005; general role in basic cellular processes,such as cell structure Basa et al.2009;Chen et al.2010).Recently,many novel maintenance and primary cellular metabolism and thus,their reference genes have been identified from Affymetrix expression is usually unaffected by external factors.An 'ideal' GeneChip data and Microarray datasets in Arabidopsis.One of reference gene for gRT-PCR has a constant and consistent the findings was that among them F-box protein(F-box),SAND expression level over all samples across different experimental family protein and mitosis protein YLS8 were more stably conditions and different tissues.However,several reports expressed than the commonly used reference genes ACTIN-2, demonstrated that there was no single gene with a constant elongation-factor-1-a (EF/-)and ubiquitin-conjugating Journal compilation CSIRO 2012 www.publish.csiro.au/joumals/fpbValidation of reference genes for real-time quantitative PCR normalisation in non-heading Chinese cabbage Dong XiaoA,B , Ning-Wen Zhang B , Jian-Jun Zhao B,C , Guusje Bonnema B,D and Xi-Lin HouA,D AState Key Laboratory of Crop Genetics and Germplasm Enhancement; Horticultural College, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China. B Laboratory of Plant Breeding, Wageningen University, The Netherlands. CHorticultural College, Hebei Agricultural University, Baoding, Hebei, China. DCorresponding author. Emails: guusje.bonnema@wur.nl; hxl@njau.edu.cn Abstract. Non-heading Chinese cabbage is an important vegetable cropthatincludes pak choi, caixin and several Japanese vegetables like mizuna, mibuna and komatsuna. Gene expression studies are frequently used to unravel the genetics of complex traits and in such studies the proper selection of reference genes for normalisation is crucial. We assessed the expression of 13 candidate reference genes includingACTIN,ACTIN-1,ACTIN-2,GAPDH, Tub_a,CyP,EF1-a, 18S rRNA, UBQ, UBC30, PPR, PP2A and MDH. Their expression stabilities were analysed using two programs, geNorm and NormFinder, in 20 different samples that represent four strategic groups. Results showed that no single gene was uniformly expressed in all tested samples. ACTIN and CyP are proposed as good reference genes when studying developmental stages. CyP, Tub_a and UBC30 are good reference genes when studying different tissues (from flowering to seed set). CyP and Tub_a are the most stable reference genes under biotic stress treatments using the fungi Peronospora parasitica and Alternaria brassicicola. UBC30, EF1-a and ACTIN are recommended for normalisation in abiotic stress studies, including hormone, salt, drought, cold and heath treatments. Moreover, at least five reference genes (ACTIN,CyP, UBC30, EF1-a and UBQ) are required for accurate qRT–PCR data normalisation when studying gene expression across all tested samples. Additional keywords: Brassica rapa ssp. chinensis, gene expression, qRT-PCR, reference genes. Received 29 October 2011, accepted 7 March 2012, published online 24 April 2012 Introduction The quantification of mRNA (mRNA) transcript levels has become an important research tool in recent years. Changes in mRNA transcript levels are crucial during plant developmental processes, between different tissues and under changing environmental conditions. Real-time quantitative PCR (qRT– PCR) has become the most popular method to quantify mRNA transcription levels and to validate whole-genome microarray data because of its outstanding accuracy, broad dynamic range and high sensitivity not only in the fields of molecular medicine, biotechnology, microbiology and molecular diagnostics but also in plant research (Vandesompele et al. 2002; Jian et al. 2008; Paolacci et al. 2009). Estimating the expression levels of target genes of interest by qRT–PCR depends on endogenous control genes to normalise qRT–PCR; control genes are also called reference genes or housekeeping genes (HKGs) (Wierschke et al. 2010; Martínez-Beamonte et al. 2011). HKGs play a general role in basic cellular processes, such as cell structure maintenance and primary cellular metabolism and thus, their expression is usually unaffected by external factors. An ‘ideal’ reference gene for qRT–PCR has a constant and consistent expression level over all samples across different experimental conditions and different tissues. However, several reports demonstrated that there was no single gene with a constant expression level under all the experimental situations tested (Kim et al. 2003; Ding et al. 2004; Argyropoulos et al. 2006). Use of inappropriate reference genes in relative quantification of gene expression profiles may lead to erroneous normalisation and consequently, misinterpretation of the results. Therefore, it is essential to validate the expression stability of reference genes in each experimental system. In plant research, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), b-ACTIN (ACTIN), tubulin a (Tub_a) and 18S rRNA were considered to have a constant expression level and as a consequence have been widely used as reference genes for normalisation of qRT–PCR data in various experimental conditions (Kim et al. 2003; Ding et al. 2004; Jian et al. 2008; Løvdal and Lillo 2009). However, it has also been reported that the transcript levels of these genes can change significantly under different experimental conditions (Czechowski et al. 2005; Terrier and Glissant et al. 2005; Basa et al. 2009; Chen et al. 2010). Recently, many novel reference genes have been identified from Affymetrix GeneChip data and Microarray datasets in Arabidopsis. One of the findings was that among them F-box protein (F-box), SAND family protein and mitosis protein YLS8 were more stably expressed than the commonly used reference genes ACTIN-2, elongation-factor-1-a (EF1-a) and ubiquitin-conjugating CSIRO PUBLISHING Functional Plant Biology, 2012, 39, 342–350 http://dx.doi.org/10.1071/FP11246 Journal compilation CSIRO 2012 www.publish.csiro.au/journals/fpb