Validation of Reliable Reference Genes for Accurate Normalization in RT-qPCR Analysis of Codonopsis pilosula
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摘要:
Abstract:
Objective To identify reliable reference genes (RGs) for normalization of real-time PCR (RT-qPCR) data in Codonopsis pilosula. Methods The expression profiles of 10 candidate RGs (GAPDH, ACT, α-TUB, β-TUB, UBQ, CYP, EF-1α, NAC, F-box and PP2A) were examined in C. pilosula during the phenological period. The raw materials examined included roots, stems, leaves, and flower buds at flowering and boll-forming stages, five growth stages of untreated and treated roots with plant growth retardant. The best-suited RGs were accessed using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Results The best-ranked references genes differed across the samples. GAPDH and PP2A were the most suitable for expression analysis in untreated tissues while GAPDH, α-TUB, and PP2A were ranked as the three most stably expressed genes in untreated roots, while NAC and CYP were the most stably expressed genes in stressed (i.e., treated) roots. The expression of UGPase, a key enzyme for CPP biosynthesis, was determined to further validate the selected RGs. Conclusion A total of 10 RGs can be used as reference genes of C. pilosula, however the appropriate one should be used as it may chance.
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National Key Technology Research and Development Program of Ministry of Science and Technology of China (2011BAI07B07); National Natural Science Foundation of China (81072987); Top Science and Technology Innovation Teams of Higher Learning Institutions of Shanxi Province (201401); Fund for Shanxi Key Subjects Construction (FSKSC) (201612)
Ling-ya Cao, Xiao-xiao Li, Dong Wang, Hai-feng Sun, Jian-ping Gao. Validation of Reliable Reference Genes for Accurate Normalization in RT-qPCR Analysis of Codonopsis pilosula[J]. Chinese Herbal Medicines (CHM),2017,9(3):226-235
