Selection of reference genes for qPCR in hairy root cultures of peanut

Abstract

BackgroundHairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments.ResultsA total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc).ConclusionsThis work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.

Reference Gene Selection for Quantitative Real-time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions

“…Similarly, two reference genes would be sufficient for the SR and PL treatments, four for the PR and CL treatments, and five for the HL treatment. In the practical application, three reference genes for the PR (V 3/4  = 0.1335) and HL (V 3/4  = 0.1480) treatments, and two for the CL treatment (V 2/3  = 0.1282) could also be accepted using a threshold value of 0.15 [14], [21], [33], [34].…”

Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper

“…This method showed that the optimal number of reference genes was 8, as indicated by the lowest Acc.S.D. (0.092); however, the setting of such a large number of reference genes is not practical [44]. Considering that Acc.S.D.…”

Enhanced Production of Resveratrol, Piceatannol, Arachidin-1, and Arachidin-3 in Hairy Root Cultures of Peanut Co-treated with Methyl Jasmonate and Cyclodextrin

“…A 96 h time course per elicitor treatment was conducted including the following time points: 0, 0.5, 1, 3, 6,9,12,15,18,21,24,30,36,40,48,72 Extraction and Analyses of Stilbenoids from the Culture Medium. Stilbenoids were extracted from the medium of NaOAc-, H 2 O 2 -, and MeJA-treated hairy roots as described before.…”