Validation of reference genes in a wide of samples Functional Plant Biology 349 heading Chinese cabbage,whereas ACTIN,CyP.UBC30,EFI-a the non-heading Chinese cabbage,analysed in this paper.In and UBO were the most stable reference genes when comparing another project conducted in our laboratory,the expression gene expression profiles of all 20 samples together. of all the 13 reference genes was tested in seven different Previously,most studies have recommended ACTIN tissue types,including whole plant of seedling,seed,stem,leaf expression as a reliable normalisation factor (Jian et al.2008) (young and old),turnip/root and flowers from two turnip However,in another study in B.rapa where expression in tissue genotypes(gene bank accessions CGN06678 and CGN07223). samples ofChinese cabbage were compared,ACTINwas not most harvested at five different developmental stages.The results stably expressed(Qi et al.2010).Jian et al.(2008)indicated that indicated that ACTIN and EF/-o.were the most stable different paralogues from the same gene family can have varying expressed genes in these tissues/developmental stages (data expression levels in different developmental stages of soybean not shown).We also conducted a gene expression profiling Our results showed that ACTIN was more stably expressed than experiment using a Nimble gene 300K array (http://www. ACTIN-/and ACTIN-2 in all four strategic groups tested and in ggbio.com/ggb/sub_contents.php?menu_id=2&sub_menu_id=0) all 20 samples analysed together.A previous observation in using 3-week-old Chinese cabbage seedlings grown under short Arabidopsis and Brachypodium distachyon showed that UBC (8 h light)or long(16 h light)daylengths with RNA extracted from had a very stable expression pattern (Czechowski et al.2005; leaves at 9 and at 21 h after dawn (see Table S1,available as Hong et al.2008).Our study also showed that the UBC30 Supplementary Material to this paper).The stability of the 13 exhibited very good expression stability when comparing its reference genes used in this study corresponded well with the expression in 20 samples and in biotic and abiotic stress results of the microarray experiment comparing growth under treated samples.The data presented in Czechowski et al. different daylengths with leaves sampled at different times of (2005),were screened for the 13 tested candidate reference the day.MDH and GAPDH were least stably expressed with genes of this study.This revealed that the transcript levels of expression levels differing at least 2-fold,whereas ACTIN and the five genes PP2A,UBO,PPR,Tub_a.and ACTIN,were very Cyp were very stable expressed similar to the results of this study. stable in developmental series and additional EF/-o was The results presented in this paper provide valuable identified as the best reference gene in abiotic stress information for future selection of reference genes in gene comparisons (Czechowski et al.2005).The EFI-a gene was expression studies in B.rapa crops,particularly non-heading also most stably expressed together with UBC30 and ACTIN in Chinese cabbage.We have identified distinct sets of genes our abiotic stress samples(Table 3).When we analysed only the appropriate for qRT-PCR in studies on plant developmental subgroup of hormone treated samples from the abiotic treatment stages,different tissues,biotic and abiotic stresses.We group,GAPDH was identified as the best ranking reference gene recommend at least five reference genes (ACTIN,Cyp, (results not shown)by both programs.Furthermore,when cold UBC30,EFI-a and UBO)for accurate gRT-PCR data and heat shock samples were analysed,GAPDH ranked first by normalisation,when studying gene expression across diverse geNorm (data not shown).However,when salt and drought types of samples.The genes ACTIN and CyP are the best choices stressed samples were analysed,GAPDH ranked last by both when studying expression in different developmental stages, programs(data not shown).This last finding is in sharp contrast whereas the genes Cyp,Tub_o.and UBC30 are preferred when with another study in heading Chinese cabbage,where GAPDH comparing gene expression in different tissues.When studying ranked as most stable reference gene during drought stress the effect ofbiotic stresses,CyP and Tub_aare recommended and (Qi et al.2010). UBC30,EF/-o and ACTINare proposed as reference genes when Recently,Hong et al.(2008)also showed that EF/-o was the studying abiotic stress.We conclude that different studies need most stably expressed gene when comparing Heat/Cold treated different sets of reference genes. Brachypodium distachyon,which was confirmed in our study by its stable expression in the abiotic stress group when analysed Acknowledgements by geNorm.Qi et al.(2010)reported that the gene CyP was not the best reference gene under drought and downy mildew stress This study was supported by the earmarked fund for the Priority Academic in Chinese cabbage.However,Cyp was identified as the most Program Development of Jiangsu Higher Education Institutions,China Agriculture Research System CARS-25-A-12 and the Fundamental stable reference gene under two temperature treatments Research Funds for the Central Universities KYZ200912.We thank (20/25C)in seagrass (Ransbotyn and Reusch 2006).In our Shuhang Wang and Dido Umer for sharing their data on expression of the study,Cyp is a stable reference gene in pakchoi for most studied gene set in turnip and yellow sarson and we thank Dr Yoonkang Hur strategic groups,except for the abiotic stress group. for performing the Nimblegen experiments. The present study indicates that none of the genes tested had a uniform expression profile in all 20 samples (tissues References developmental stages,stress responses),which stresses the fact that more reference genes need to be included when diverse sets Andersen CL,Jensen JL,Orntoft TF (2004)Normalization of real-time of samples are compared,as suggested by the geNorm software. quantitative reverse transcription-PCR data:a model-based variance Thus,before studying gene expression by qRT-PCR,it is estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets.Cancer Research 64(15). necessary to consider a suitable set of reference genes. 5245-5250.do:10.1158/0008-5472.CAN-04-0496 We also tested these genes in different B.rapa accessions Argyropoulos D,Psallida C,Spyropoulos CG(2006)Generic normalization that represent different morpho types.Both in yellow sarson method for real-time PCR:application for the analysis of the mannanase (annual oil crop)and fodder tumip,the primers amplified PCR gene expressed in germinating tomato seed.FEBS Journal 273(4). products with similar efficiency(data not shown)compared with 770-777.doi:10.1111万.1742-4658.2006.05109.xheading Chinese cabbage, whereas ACTIN, CyP, UBC30, EF1-a and UBQ were the most stable reference genes when comparing gene expression profiles of all 20 samples together. Previously, most studies have recommended ACTIN expression as a reliable normalisation factor (Jian et al. 2008). However, in another study in B. rapa where expression in tissue samples ofChinese cabbage were compared,ACTINwas not most stably expressed (Qi et al. 2010). Jian et al. (2008) indicated that different paralogues from the same gene family can have varying expression levels in different developmental stages of soybean. Our results showed that ACTIN was more stably expressed than ACTIN-1 and ACTIN-2 in all four strategic groups tested and in all 20 samples analysed together. A previous observation in Arabidopsis and Brachypodium distachyon showed that UBC had a very stable expression pattern (Czechowski et al. 2005; Hong et al. 2008). Our study also showed that the UBC30 exhibited very good expression stability when comparing its expression in 20 samples and in biotic and abiotic stress treated samples. The data presented in Czechowski et al. (2005), were screened for the 13 tested candidate reference genes of this study. This revealed that the transcript levels of the five genes PP2A, UBQ, PPR, Tub_a and ACTIN, were very stable in developmental series and additional EF1-a was identified as the best reference gene in abiotic stress comparisons (Czechowski et al. 2005). The EF1-a gene was also most stably expressed together with UBC30 and ACTIN in our abiotic stress samples (Table 3). When we analysed only the subgroup of hormone treated samples from the abiotic treatment group, GAPDH was identified as the best ranking reference gene (results not shown) by both programs. Furthermore, when cold and heat shock samples were analysed, GAPDH ranked first by geNorm (data not shown). However, when salt and drought stressed samples were analysed, GAPDH ranked last by both programs (data not shown). This last finding is in sharp contrast with another study in heading Chinese cabbage, where GAPDH ranked as most stable reference gene during drought stress (Qi et al. 2010). Recently, Hong et al. (2008) also showed that EF1-a was the most stably expressed gene when comparing Heat/Cold treated Brachypodium distachyon, which was confirmed in our study by its stable expression in the abiotic stress group when analysed by geNorm. Qi et al. (2010) reported that the gene CyP was not the best reference gene under drought and downy mildew stress in Chinese cabbage. However, CyP was identified as the most stable reference gene under two temperature treatments (20/25
C) in seagrass (Ransbotyn and Reusch 2006). In our study, CyP is a stable reference gene in pakchoi for most strategic groups, except for the abiotic stress group. The present study indicates that none of the genes tested had a uniform expression profile in all 20 samples (tissues, developmental stages, stress responses), which stresses the fact that more reference genes need to be included when diverse sets of samples are compared, as suggested by the geNorm software. Thus, before studying gene expression by qRT–PCR, it is necessary to consider a suitable set of reference genes. We also tested these genes in different B. rapa accessions that represent different morpho types. Both in yellow sarson (annual oil crop) and fodder turnip, the primers amplified PCR products with similar efficiency (data not shown) compared with the non-heading Chinese cabbage, analysed in this paper. In another project conducted in our laboratory, the expression of all the 13 reference genes was tested in seven different tissue types, including whole plant of seedling, seed, stem, leaf (young and old), turnip/root and flowers from two turnip genotypes (gene bank accessions CGN06678 and CGN07223), harvested at five different developmental stages. The results indicated that ACTIN and EF1-a were the most stable expressed genes in these tissues/developmental stages (data not shown). We also conducted a gene expression profiling experiment using a Nimble gene 300K array (http://www. ggbio.com/ggb/sub_contents.php?menu_id=2&sub_menu_id=0) using 3-week-old Chinese cabbage seedlings grown under short (8 h light) or long (16 h light) daylengths with RNA extracted from leaves at 9 and at 21 h after dawn (see Table S1, available as Supplementary Material to this paper). The stability of the 13 reference genes used in this study corresponded well with the results of the microarray experiment comparing growth under different daylengths with leaves sampled at different times of the day. MDH and GAPDH were least stably expressed with expression levels differing at least 2-fold, whereas ACTIN and Cyp were very stable expressed similar to the results of this study. The results presented in this paper provide valuable information for future selection of reference genes in gene expression studies in B. rapa crops, particularly non-heading Chinese cabbage. We have identified distinct sets of genes appropriate for qRT–PCR in studies on plant developmental stages, different tissues, biotic and abiotic stresses. We recommend at least five reference genes (ACTIN, CyP, UBC30, EF1-a and UBQ) for accurate qRT–PCR data normalisation, when studying gene expression across diverse types of samples. The genes ACTIN and CyP are the best choices when studying expression in different developmental stages, whereas the genes CyP, Tub_a and UBC30 are preferred when comparing gene expression in different tissues. When studying the effect of biotic stresses,CyPand Tub_aare recommended and UBC30,EF1-aandACTINare proposed as reference genes when studying abiotic stress. We conclude that different studies need different sets of reference genes. Acknowledgements This study was supported by the earmarked fund for the Priority Academic Program Development of Jiangsu Higher Education Institutions, China Agriculture Research System CARS-25-A-12 and the Fundamental Research Funds for the Central Universities KYZ200912. We thank Shuhang Wang and Dido Umer for sharing their data on expression of the studied gene set in turnip and yellow sarson and we thank Dr Yoonkang Hur for performing the Nimblegen experiments. References Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64(15), 5245–5250. doi:10.1158/0008-5472.CAN-04-0496 Argyropoulos D, Psallida C, Spyropoulos CG (2006) Generic normalization method for real-time PCR: application for the analysis of the mannanase gene expressed in germinating tomato seed. FEBS Journal 273(4), 770–777. doi:10.1111/j.1742-4658.2006.05109.x Validation of reference genes in a wide of samples Functional Plant Biology 349