Selection and validation of reference genes for RT-qPCR normalization in different tissues of milk thistle (Silybum marianum, Gaert.)

Milk thistle is grown in the Czech Republic as a medicinal herb; silymarin is isolated from its achenes and used for the production of liver and gallbladder medicine. The quality and content of the active compound is influenced not only by environmental factors, but also by pests and pathogens. The occurrence of pests of milk thistle variety Silyb was observed in two localities during the years 2011 2013. In the year 2011 the mycoflora of seeds of four milk thistle varieties was determined. Representatives of 15 species were isolated from the seeds, most of them saprophytic. 21 fungal species were isolated and identified from milk thistle plants during the vegetation; Septoria silybi among the most important ones. Possibilities of protection of milt thistle against pathogens are discussed.

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A COMPARATIVE STUDY OF STANDARDIZATION METHODS FOR THE MILK THISTLE FRUIT (SILYBUM MARIANUM)

University proceedings Volga region Medical sciences ◽

10.21685/2072-3032-2016-4-4 ◽

2016 ◽

Author(s):

S. A. Burkush ◽

◽

S. V. Chizhov ◽

N. V. Firstova ◽

N. N. Glebova ◽

...

Keyword(s):

Comparative Study ◽

Milk Thistle ◽

Silybum Marianum ◽

Standardization Methods

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Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

Scientific Reports ◽

10.1038/s41598-020-79298-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuping Li ◽

Xiaoju Liang ◽

Xuguo Zhou ◽

Yu An ◽

Ming Li ◽

...

Keyword(s):

Gene Expression ◽

Developmental Stage ◽

Reference Genes ◽

Developmental Stages ◽

Individual Factors ◽

Congeneric Species ◽

Spatio Temporal ◽

The Stability ◽

Different Tissues ◽

Selection Of

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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Selection and optimization of reference genes for RT-qPCR normalization: A case study in Solanum lycopersicum exposed to UV-B

Plant Physiology and Biochemistry ◽

10.1016/j.plaphy.2021.01.026 ◽

2021 ◽

Vol 160 ◽

pp. 269-280

Author(s):

María Belén Fernández ◽

Germán Lukaszewicz ◽

Lorenzo Lamattina ◽

Raúl Cassia

Keyword(s):

Solanum Lycopersicum ◽

Reference Genes ◽

Qpcr Normalization

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Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone Flaccida Fr. Schmidt at Different Growing Stages

Genes ◽

10.3390/genes12030459 ◽

2021 ◽

Vol 12 (3) ◽

pp. 459

Author(s):

Zeying Zhao ◽

Hanwen Zhou ◽

Zhongnan Nie ◽

Xuekui Wang ◽

Biaobiao Luo ◽

...

Keyword(s):

Reference Genes ◽

Gene Selection ◽

Quantitative Polymerase Chain Reaction ◽

Cultivated Plants ◽

Gene Expressions ◽

Natural Habitats ◽

Triterpenoid Saponins ◽

Qpcr Normalization ◽

Suitable Reference

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

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Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca’s response to salinity

Scientific Reports ◽

10.1038/s41598-021-88151-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Meng Wang ◽

Tingting Ren ◽

Prince Marowa ◽

Haina Du ◽

Zongchang Xu

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Suitable Reference Gene ◽

Normalize Gene Expression ◽

Suaeda Glauca ◽

Suitable Reference ◽

Germinating Seeds ◽

Different Tissues

AbstractQuantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

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Protective Effects of Milk Thistle (Silybum marianum) against Aflatoxin B1 in Broiler Chicks

Asian-Australasian Journal of Animal Sciences ◽

10.5713/ajas.2011.10418 ◽

2011 ◽

Vol 24 (7) ◽

pp. 1011-1018 ◽

Cited By ~ 18

Author(s):

N. Chand ◽

Din Muhammad ◽

F. R. Durrani ◽

M. Subhan Qureshi ◽

Sahibzada S Ullah

Keyword(s):

Aflatoxin B1 ◽

Milk Thistle ◽

Broiler Chicks ◽

Protective Effects ◽

Silybum Marianum

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Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

10.21203/rs.3.rs-145449/v1 ◽

2021 ◽

Author(s):

Zhongyi Yang ◽

Rui Zhang ◽

Zhichun Zhou

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Geometric Mean ◽

Gene Transcript ◽

Expression Stability ◽

Schima Superba ◽

Expression Studies ◽

Gene Expression Studies ◽

Different Tissues

Abstract Background Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

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GERMINATION AND SEEDLING GROWTH OF SILYBUM MARIANUM AS A MEDICINAL PLANT UNDER SALINITY STRESS

Plant Breeding and Seed Science ◽

10.37317/pbss-2019-0003 ◽

2019 ◽

pp. 25-38

Author(s):

M. A. Dorri ◽

B. Kamkar ◽

M. Aghdasi ◽

A. R. Safahani

Keyword(s):

Medicinal Plant ◽

Seedling Growth ◽

Block Design ◽

Germination Percentage ◽

Milk Thistle ◽

Tolerance Index ◽

Root Weight ◽

Silybum Marianum ◽

Nacl Concentration ◽

Salinity Levels

Milk thistle (Silybum marianum) is used as a medicinal plant in the treatment of liver diseases. In order to investigate germination and seedling growth in S. marianum subjected to NaCl, a three replicated experiment was carried out in a randomized complete block design in the laboratory of Gorgan University of Agricultural Sciences and Natural Resources. Treatments included seven salinity levels (50, 75, 100, 125, 150, 175 and 200 mM) and a control (distilled water). Germination percentage (GP), mean germination time (MGT), seed-ling vigour index (SVI), shoot, root and seedling weight were measured. The effect of salinity levels was significant on GP, SVI, MGT and seedling weight and length (p ≤ 0.01). However, the NaCl concentration effect was not significant on shoot : root length ratio and or shoot : root weight ratios. Results showed that germination decreased when salinity increased, while MGT increased. MGT was 1.75 times higher than in the control at the highest salt concentration. MGT difference was not significant between 75 mM NaCl and con-trol, while it dramatically increased by increasing the NaCl concentration from 150 to 200 mM NaCl. Reduc-tion slope and salt tolerance index (STI) were estimated for germination (0.54 and 231.9) and seedling stage (0.24 and 237.4). According to the results, milk thistle could be considered as a valuable medicinal plant in fairly salinized areas.

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Selecting Suitable Reference Genes for qPCR Normalization: A Comprehensive Analysis in MCF-7 Breast Cancer Cell Line

10.21203/rs.3.rs-42293/v2 ◽

2020 ◽

Author(s):

Nityanand Jain ◽

Dina Nitisa ◽

Valdis Pirsko ◽

Inese Cakstina

Keyword(s):

Gene Expression ◽

Cell Line ◽

Reference Gene ◽

Reference Genes ◽

Cancer Cell Line ◽

Internal Controls ◽

Reference Gene Expression ◽

Qpcr Normalization ◽

Suitable Reference ◽

Mcf 7

Abstract BackgroundMCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study have not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to devise an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies.ResultsThe analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference gene pair while for culture A2, GAPDH-RNA28S was identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-PCBP1-CCSER2 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress.ConclusionsThe variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

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