Reference gene selection for qRT-PCR in Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] as affected by various abiotic factors
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The determination of the best reference gene is essential to improve and guarantee the accuracy of the qPCR technique. Thus, the objective of this work was to evaluate the normalization genes efficiency for qRT-PCR studies of Pfaffia glomerata, a species with marked medicinal and industrial interests due to the production of the phytoecdysteroid 20-hydroxyecdysone (20-E). We have selected four candidates as reference genes in P. glomerata: elongation factor-1α (EF-1α), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), spectrin-like (SPT) and α-tubulin (TUA), and tested their expression stabilities as affected by abiotic factors (salt stress, drought stress, irradiance, photoperiod and CO2 enrichment), using the following methods: NormFinder, geNorm, BestKeeper, ΔCt and RefFinder. Also, the Phantom gene, which belongs to the 20-E biosynthesis pathway, was targeted to validate the most stable reference gene for each abiotic factor. The GAPDH was the most stable gene under all assessed abiotic factors, as well as the most stable when data from all experiments were taken together, which was confirmed by all the software used. The use of more or less stable reference genes for normalization significantly changes the interpretation of the qPCR data, evidencing the importance of choosing the most appropriate housekeeping gene for each expression assay. Based on our results, we recommend GAPDH to be used for normalization of qPCR expression data in P. glomerata in diverse abiotic conditions. This work is the first report on the validation of reference genes in P. glomerata and will be fundamental for further gene expression studies in this important medicinal species.
GAPDH is the most stable gene to be used for the normalization in qPCR analyzes in [Pfaffia glomerata (Spreng.) Pedersen] under the different abiotic factors.
KeywordsAbiotic conditions Brazilian ginseng Gene expression Quantitative real time Reference gene
The authors thank the Brazilian sponsoring agencies, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior), for financial support.
DSB, SHSF, EAF, TDS, KC and EL conceived, designed and performed the experiments; DSB and VSM collected and analyzed the data; DSB, VSM, MGCC, and WCO contributed to the design and interpretation of the research and to the writing of the paper. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of realtime quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250. https://doi.org/10.1158/0008-5472.CAN-04-0496 CrossRefGoogle Scholar
- Artico S, Nardeli SM, Brilhante O, Grossi-de-Sa MF, Alves-Ferreira M (2010) Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biol 10:49. https://doi.org/10.1186/1471-2229-10-49 CrossRefGoogle Scholar
- Batista DS, Koehler AD, Romanel E, Souza VC, Silva TD, Almeida MC, Maciel TEF, Ferreira PRB, Felipe SHS, Saldanha CW, Maldaner J, Dias LLC, Festucci-Buselli RA, Otoni WC (2018) De novo assembly and transcriptome of Pfaffia glomerata uncovers the role of photoautotrophy and the P450 family genes in 20-hydroxyecdysone production. Protoplasma. https://doi.org/10.1007/s00709-018-1322-1 Google Scholar
- Corrêa JPO, Vital CE, Pinheiro MVM, Batista DS, Azevedo JFL, Saldanha CW, Cruz ACF, DaMatta FM, Otoni WC (2015) In vitro photoautotrophic potential and ex vitro photosynthetic competence of Pfaffia glomerata (Spreng.) Pedersen accessions. Plant Cell Tissue Organ Cult 121:289–300. https://doi.org/10.1007/s11240-014-0700-4 CrossRefGoogle Scholar
- Corrêa JPO, Vital CE, Pinheiro MVM, Batista DS, Saldanha CW, Cruz ACF, Notini MM, Freitas DMS, DaMatta FM, Otoni WC (2016) Induced polyploidization increases 20-hydroxyecdysone content, in vitro photoautotrophic growth, and ex vitro biomass accumulation in Pfaffia glomerata (Spreng.) Pedersen. Vitro Cell Dev Biol Plant 52:45–55. https://doi.org/10.1007/s11627-016-9746-9 CrossRefGoogle Scholar
- De Keyser E, Desmet L, Van Bockstaele E, De Riek J (2013) How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population. BMC Mol Biol 14:13. https://doi.org/10.1186/1471-2199-14-13 CrossRefGoogle Scholar
- De Spiegelaere W, Dern-Wieloch J, Weigel R, Schumacher V, Schorle H, Nettersheim D, Bergmann M, Brehm R, Kliesch S, Vandekerckhove L, Fink C (2015) Reference gene validation for RT-qPCR, a note on different available software packages. PLoS ONE 10:e0122515. https://doi.org/10.1371/journal.pone.0122515 CrossRefGoogle Scholar
- Dohm JC, Minoche AE, Holtgrawe D, Capella-Gutierrez S, Zakrzewski F, Tafer H, Rupp O, Sorensen TR, Stracke R, Reinhardt R, Goesmann A, Kraft T, Schulz B, Stadler PF, Schmidt T, Gabaldón T, Lehrach H, Weisshaar B, Himmelbauer H (2014) The genome of the recently domesticated crop plant sugar beet (Beta vulgaris). Nature 505:546–549. https://doi.org/10.1038/nature12817 CrossRefGoogle Scholar
- Gutierrez L, Mauriat M, Guénin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6:609–618. https://doi.org/10.1111/j.1467-7652.2008.00346.x CrossRefGoogle Scholar
- Moreira VS, Soares VL, Silva RJ, Sousa AO, Otoni WC, Costa MG (2018) Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L. Physiol Mol Biol Plants 24:369–378. https://doi.org/10.1007/s12298-018-0528-1 CrossRefGoogle Scholar
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
- Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515. https://doi.org/10.1023/B:BILE.0000019559.84305.47 CrossRefGoogle Scholar
- Saldanha CW, Otoni CG, Notini MM, Kuki KN, Cruz ACF, Rubio Neto A, Dias LLC, Otoni WC (2013) A CO2-enriched atmosphere improves in vitro growth of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Vitro Cell Dev Biol Plant 49:433–444. https://doi.org/10.1007/s11627-013-9529-5 CrossRefGoogle Scholar
- Saldanha CW, Otoni CG, Rocha DI, Cavatte PC, Detmann KDSC, Tanaka FAO, Dias LLC, DaMatta FM, Otoni WC (2014) CO2-enriched atmosphere and supporting material impact the growth, morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets. Plant Cell, Tissue Organ Cult 118:87–99. https://doi.org/10.1007/s11240-014-0464-x CrossRefGoogle Scholar
- Sinha P, Singh VK, Suryanarayana V, Krishnamurthy L, Saxena RK, Varshney RK (2015) Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions. PLoS ONE 10:e0122847. https://doi.org/10.1371/journal.pone.0122847 CrossRefGoogle Scholar
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(research0034):1. https://doi.org/10.1186/gb-2002-3-7-research0034 Google Scholar
- Zhang Y, Han X, Chen S, Zheng L, He X, Liu M, He X, Liu M, Qiao G, Wang Y, Zhuo R (2017) Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses. Sci Rep 7:40290. https://doi.org/10.1038/srep40290 CrossRefGoogle Scholar