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Open Access Highly Accessed Research article

Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say)

Xiao-Qin Shi1, Wen-Chao Guo2, Pin-Jun Wan1, Li-Tao Zhou1, Xiang-Liang Ren1, Tursun Ahmat2, Kai-Yun Fu1 and Guo-Qing Li1*

Author Affiliations

1 Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

2 Department of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

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BMC Research Notes 2013, 6:93  doi:10.1186/1756-0500-6-93

Published: 13 March 2013

Abstract

Background

L. decemlineata is an exotic invasive insect pest, and invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan. It is a notorious defoliator of potato throughout most of the northern Xinjiang in current, and often causes extremely large yield losses of potato.

Results

The expression stability of nine L. decemlineata house-keeping genes (Actin, ACT1 and ACT2; ADP-ribosylation factor, ARF1 and ARF4; TATA box binding protein, TBP1 and TBP2; ribosomal protein RP4 and RP18; translation elongation factor 1α EF1α) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in seven developmental stages, three larval tissues and two insecticide treatments. The results were analyzed using three software programs: geNorm, NormFinder and BestKeeper. Although there was no consistent ranking observed among the house-keeping genes across the samples, the overall analysis revealed that RP18, RP4, ARF1, and ARF4 were the four most stable house-keeping genes. In contrast, ACT1 and ACT2, two of the most widely used reference genes, had the least stability. Our results suggest that the combined use of the four most stably expressed genes may produce optimal normalization for qRT-PCR.

Conclusions

The expression stability of the house-keeping genes varies among different developing stages, in different tissues and under different experimental conditions. Our results will enable a more accurate and reliable normalization of qRT-PCR data in L. decemlineata.

Keywords:
L. decemlineata; Quantitative real-time PCR; Reference gene; Normalization