selection and validation of endogenous reference genes for qrt-pcr analysis in leafy spurge (euphorbia esula)选择和验证内生参考基因中存在分析叶大戟(大戟属植物esula).pdfVIP

Selection and Validation of Endogenous Reference Genes for qRT-PCR Analysis in Leafy Spurge (Euphorbia esula) ¨ Wun S. Chao*, Munevver Dog˘ ramaci, Michael E. Foley, David P. Horvath, James V. Anderson United States Department of Agriculture-Agricultural Research Service, Biosciences Research Lab, Sunflower and Plant Biology Research Unit, Fargo, North Dakota, United States of America Abstract Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis ‘‘general purpose’’ traditional reference genes (GAPDH_1, GAPDH_2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the CT values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative DCT metho