Research Article Open Access

Genome-Wide Selection and Testing of Superior Reference Genes for Quantitative Gene Expression Normalization in Tobacco (Nicotiana tabacum)

Abstract

qRT-PCR is a very accurate tool for the measurement of gene expression in all organisms. Accurate and reproducible qRT-PCR results depend on the normalization of expression data using reliable reference genes. To diversify the pool of reliable reference genes in tobacco, the expression stability of 44,873 tobacco genes (ESTs) were measured using a custom-designed microarray in a diverse set of 21 samples, representing various tissue types and developmental stages. A total of 14 genes were selected as candidate reference genes because they appeared to be the most stable. These genes were compared with two widely used housekeeping genes (L25 and EF-1a) for further validation by qRT-PCR. The results showed that Gene 3 (NADH dehydrogenase) and Gene 4 (chaperonin CPN60-2) exhibited the highest expression stability and that they were even more stable than L25 and EF-1a. Taken together, our results provide a wider pool of stable reference genes for gene expression analysis of tobacco.

Yushuang Guo, Ruiyuan Li, Zeshan Asgher, Imran Haider Shamsi, Shizhou Yu, Jiehong Zhao, and Xueliang Ren

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