Back to Contents

Pak. J. Bot., 47(5): 1793-1802, 2015.

FREE FULL TEXT PDF

EXPLORING VALID REFERENCE GENES FOR QUANTITATIVE REAL-TIME RT-PCR STUDIES OF HYDROGEN PEROXIDE SIGNALING IN ARABIDOPSIS

HENG ZHOU, BIN HAN, YANJIE XIE, JING ZHANG AND WENBIAO SHEN^*

Abstract: Hydrogen peroxide (H₂O₂) acts as a signaling molecule modulating the expression of various genes in plants. However, the reference gene(s) used for gene expression analysis of H₂O₂ signaling is still arbitrary. A reliable result obtained by quantitative real-time RT-PCR (RT-qPCR) highly depends on accurate transcript normalization using stably expressed reference genes, whereas the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated and compared in root and shoot tissues of Arabidopsis upon different doses of H₂O₂. The results revealed that, in our experimental conditions, three novel reference genes (TIP41-like, UKN, and UBC21) were identified and validated as suitable reference genes for RT-qPCR normalization in both root and shoot tissues under oxidative stress. This conclusion was further confirmed by publicly available microarray data of methyl viologen and drought stress. In comparison with a single reference gene (EF-1a), the expression pattern of ZAT12 modulated by H₂O₂, when using TIP41-like, UKN, and UBC21 as multiple reference gene(s), was similar with the previous reports by using northern blotting. Thus, we proposed that these three reference genes might be good candidates for other researchers to include in their reference gene validation in gene expression studies under H₂O₂-related oxidative stress.

Key words: Arabidopsis, Quantitative real-time RT-PCR, geNorm, NormFinder algorithms.

College of Life Sciences, Laboratory Center of Life Science, Nanjing Agricultural University, Nanjing 210095, China

*Correspondence author’s e-mail: wbshenh@njau.edu.cn