Selection and validation of reference genes for quantitative real-time PCR in Rosmarinus officinalis L. in various tissues and under elicitation

Aminfar, Zahra; Rabiei, Babak; Tohidfar, Masoud; Mirjalili, Mohammad Hossein

doi:10.1016/j.bcab.2019.101246

Cited by 10 publications

(11 citation statements)

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“…Although we had identified and validated reference genes suitable for normalizing RT-qPCR experiments in in vitro-produced rosemary, there are no internal reference genes that are stably expressed under all experimental conditions. For example, in a previous study with Rosmarinus officinalis , the authors recommend using GAPDH for normalization of the RT-qPCR data [ 13 ], but the current study suggests that this gene may be unsuitable for expression studies with plant tissue culture-produced rosemary. These contradictory differences might be attributed to plant tissue selection, different stress treatments and experimental treatments carried out under in vitro conditions.…”

Section: Discussionmentioning

confidence: 99%

“…Hence, this study aims to identify a suitable reference gene for gene expression studies in in vitro S. rosmarinus . We selected and identified seven common candidate reference genes ( 18S rRNA, 25S rRNA, 28S rRNA, ACCase, GAPDH, ATP-synthase, and F1-ATPase ) from previous literature studies [ 7 , 8 , 13 , 14 ]. The genes identified were from S. rosmarinus , related or other plant species depending on their homology, and how frequently are utilized.…”

Section: Introductionmentioning

confidence: 99%

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Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia rosmarinus under In Vitro Conditions

Bharati

Sen

Kumar

et al. 2022

Plants

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Salvia rosmarinus L. (rosemary) is known to have a wide range of pharmacological effects including antidiabetic, anticarcinogenic, and antitumorigenic properties owing to its secondary metabolites. Studies aiming to elevate these metabolites have utilized various elicitors and stresses under in vitro conditions, although underlying molecular mechanisms remain unexplored. Gene expression studies using RT-qPCR might provide valuable information regarding how plant and plant cells interact and perceive various treatments and elicitors. However, despite being able to calculate accurate fold changes, the accuracy of the RT-qPCR data highly depends on the expression of reference genes. To the best of our knowledge, there is no information available on the stable reference genes in rosemary under in vitro conditions. Thus, in this paper, we assessed the stability of seven commonly used reference genes under different elicitor and stress conditions using RT-qPCR. Thereafter, the five most commonly used software and algorithms (comparative ΔCt, BestKeeper, NormFinder, geNorm, and RefFinder) were used to rank the candidates based on their expression stabilities. In conclusion, we recommend using a combination of F1-ATPase, ATP synthase and ACCase to normalize the gene expression experiments in rosemary under in vitro conditions. The selected reference genes were verified using 4-coumarate-CoA ligase, a pharmacologically important gene, whose expression might alter under nanoparticle treatment. Additionally, reference genes for several plant tissues, elicitors, and stresses are also proposed. The conclusions obtained from this current study will accelerate the future molecular work in S. rosmarinus and other related species.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia rosmarinus under In Vitro Conditions

Bharati

Sen

Kumar

et al. 2022

Plants

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“…EF-1α expression was also assessed in green microalgae such as Volvox carteri [ 55 ] and Chlamydomonas [ 17 , 57 ], although since only EFL has been reported in green algae it seems likely that the putative EF-1α was misannotated [ 66 , 83 ]. In addition, the inclusion in the present study of KAS as a potential reference gene for normalization is new to microalgae, and came from previous data about gene stability in the microalgae Nannochloropsis gaditana [ 84 ]. Finally, the remaining genes ( bTUB , PGK , His2A , rbcL , cdkA , UBCE , ALD , RPL32 and RPS10 ) were selected as they have been previously tested as reference genes in microalgae.…”

Section: Discussionmentioning

confidence: 99%

“…Several statistical algorithms have been developed to identify the most suitable reference genes under a given set of experimental or biological conditions in order to avoid artifacts due to normalization, and include geNorm [ 27 ], NormFinder [ 28 ], BestKeeper [ 29 ], the comparative Δ C T method [ 30 ], and the web-based program RefFinder [ 31 ]. These tools have been carried out on a broad range of species, including whole organisms, specific tissues or cell types from animals [ 19 – 21 , 32 , 33 ], plants [ 34 – 37 ], and fungi [ 38 , 39 ]. Selection of reference genes has also been addressed in macroalgae [ 40 – 42 ] as well as in specific microalgae species, including the diatoms Phaeodactylum tricornutum [ 43 ], Pseudo-nitzschia multristrata and P .…”

Section: Introductionmentioning

confidence: 99%

Selection and validation of reference genes for quantitative real-time PCR in the green microalgae Tetraselmis chui

Torres¹,

Lama²,

Mantecón³

et al. 2021

PLoS ONE

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Quantitative real-time reverse transcription PCR (RT-qPCR) is a highly sensitive technique that can be applied to analyze how genes are modulated by culture conditions, but identification of appropriate reference genes for normalization is a critical factor to be considered. For this reason, the expression stability of 18 candidate reference genes was evaluated for the green microalgae Tetraselmis chui using the widely employed algorithms geNorm, NormFinder, BestKeeper, the comparative ΔCT method, and RefFinder. Microalgae samples were collected from large scale outdoor photobioreactors during the growing phase (OUT_GP), and during the semi-continuous phase at different times of the day (OUT_DC). Samples from standard indoor cultures under highly controlled conditions (IND) were also collected to complement the other data. Different rankings for the candidate reference genes were obtained depending on the culture conditions and the algorithm employed. After comparison of the achieved ranks with the different methods, the references genes selected for samples from specific culture conditions were ALD and EFL in OUT_GP, RPL32 and UBCE in OUT_DC, and cdkA and UBCE in IND. Moreover, the genes EFL and cdkA or EFL and UBCE appeared as appropriate combinations for pools generated from all samples (ALL). Examination in the OUT_DC cultures of genes encoding the large and small subunits of ADP-glucose pyrophosphorylase (AGPL and AGPS, respectively) confirmed the reliability of the identified reference genes, RPL32 and UBCE. The present study represents a useful contribution for studies of gene expression in T. chui, and also represents the first step to set-up an RT-qPCR platform for quality control of T. chui biomass production in industrial facilities.

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“…The grapevine is a plant with different tissues, including fruits, leaves, tendrils, roots and flowers. It is necessary to evaluate the stability of reference genes in different tissues, so that we can choose specific reference genes for samples of specific tissues (Nicholson et al 2012, Aminfar et al 2019). The development of the berry is an important trait of vines, and studies on its developmental stages are increasingly significant (Ollat et al 2002).…”

Section: Discussionmentioning

confidence: 99%

Identification of optimal and novel reference genes for quantitative real‐time polymerase chain reaction analysis in grapevine

Wei

Wang

Liu

et al. 2021

Australian Journal of Grape and Wine Research

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Background and Aims Quantitative real‐time polymerase chain reaction (qRT‐PCR) has the advantages of sensitivity, specificity and repeatability, and is commonly used to investigate the expression levels of genes. The selection of appropriate endogenous reference genes is the pre‐condition for acquiring the desired results of qRT‐PCR. This study aimed to identify and screen the most suitable reference genes for grapevines. Methods and Results Three novel genes from previous transcriptomic data were selected for their stable expression either at the developmental stages of the grape berry or under the various treatments. The expression stability of these three candidate genes and six commonly used reference genes were evaluated with geNorm, NormFinder and BestKeeper algorithms in several grapevine samples. The geNorm analysis indicated that two reference genes were sufficient to normalise expression. The optimal reference gene combinations were: VvEF1‐γ and PPR2 in fruit; RRM1 and EF1‐α in leaf; EF1‐α and Actin in tendril of several cultivars. The combination of EF1‐α and VvEF1‐α was the most appropriate reference gene for fruits at different developmental stages. When all the samples were considered, the combination of RRM1 and Ubiquitin was optimal. Conclusions Different reference genes should be employed when qRT‐PCR is performed in different grapevine samples. The gene RRM1 is a new appropriate reference gene for grapevine. Significance of the Study This study provided alternatives for the optimal reference genes in qRT‐PCR, and identified one novel and appropriate reference gene (RRM1) for grapevine.

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Selection and validation of reference genes for quantitative real-time PCR in Rosmarinus officinalis L. in various tissues and under elicitation

Cited by 10 publications

References 76 publications

Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia rosmarinus under In Vitro Conditions

Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia rosmarinus under In Vitro Conditions

Selection and validation of reference genes for quantitative real-time PCR in the green microalgae Tetraselmis chui

Identification of optimal and novel reference genes for quantitative real‐time polymerase chain reaction analysis in grapevine

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