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Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Anoectochilus roxburghii

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Abstract

Accurate quantification of transcript profiling with quantitative real time polymerase chain reaction (qRT-PCR) relies on the reliable normalization of an appropriate reference gene. This study reported the identification and validation of nine reference genes, including β-tubulin (β-TUB), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), actin 1/2(ACT-1 and ACT-2), 18S rRNA, and 26S rRNA, from Anoectochilus roxburghii (Wall.) Lindl., a valuable herb remedy widely used for various diseases treatment in traditional Chinese medicine. Transcriptional levels of the candidate reference genes were examined using qRT-PCR analysis and revealed differential expression of the genes in the leaf, stem, root, flower, and peduncle tissues. The relative quantities data were subjected to geNorm software for ranking the expression stability of the reference genes and the results showed that EF-1β and ACT-2 were the two best stable genes whereas GAPDH and 26S rRNA did not favor normalization of qRT-PCR in these tissues. The expression pattern of a squalene synthase encoding gene (SS) was also determined in parallel. The analyses were in great consistency when the qRT-PCR data was normalized to the expression of each or both of EF-1β and ACT-2 as the internal control, further confirming the reliability of EF-1β and ACT-2 as the best internal control. The present study provided the first important clues for accurate data normalization in transcript profiling in A. roxburghii, which will be essential to further functional genomics study in the valuable medicinal plant.

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Abbreviations

ACT-1:

Actin 1

ACT-2:

Actin 2

CT:

Threshold value

E :

PCR efficiency

EF-1α :

Elongation factor 1 alpha

EF-1β :

Elongation factor 1 beta

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

Q :

Relative quantities

qRT-PCR:

Quantitative reverse transcription PCR

RT-PCR:

Reverse transcription polymerase chain reaction

sqPCR:

Semi-quantitative PCR

UBQ:

Ubiquitin

18S rRNA:

18S ribosomal RNA

26S rRNA:

26S ribosomal RNA

β-TUB:

β-Tubulin

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Acknowledgments

This research was financially supported by the National Natural Sciences Foundation of China (No. 31101608 and 31070300), and the China Postdoctoral Science Foundation (No. 20100470244).

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Authors and Affiliations

  1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193, China

    Gang Zhang, Mingming Zhao, Chao Song, Anxiong Luo & Shunxing Guo

  2. College of Pharmacy and Shaanxi Provincial Key Laboratory for Chinese Medicine Basis & New Drugs Research, Shaanxi University of Chinese Medicine, Xi’an, Shaanxi, 712046, China

    Gang Zhang

  3. College of Veterinary Medicine, Kansas State University, L-222 Mosier Hall, Manhattan, KS, 66506, USA

    Jianfa Bai

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  1. Gang Zhang
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  2. Mingming Zhao
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Correspondence to Shunxing Guo.

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Zhang, G., Zhao, M., Song, C. et al. Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Anoectochilus roxburghii . Mol Biol Rep 39, 5905–5912 (2012). https://doi.org/10.1007/s11033-011-1402-1

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