Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation

被引:18
作者
Basa, Brigitta [1 ]
Solti, Adam [1 ]
Sarvari, Eva [1 ]
Tamas, Laszlo [1 ]
机构
[1] Eotvos Lorand Univ, Dept Plant Physiol & Mol Plant Biol, H-1117 Budapest, Hungary
关键词
Populus; qRT-PCR; reference gene; RiboGreen; validation; PROTEASOME PROTEOLYTIC PATHWAY; HEAVY-METAL STRESS; RT-PCR; REVERSE TRANSCRIPTION; ARABIDOPSIS-THALIANA; RESPONSIVE GENES; QUANTITATIVE-PCR; INTERNAL CONTROL; PHOTOSYSTEM-II; CADMIUM;
D O I
10.1071/FP09073
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Real-time RT-PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quanti. cation the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1 alpha, polyubiquitin, tubulin beta-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quanti. cation method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1 alpha, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.
引用
收藏
页码:1079 / 1087
页数:9
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