scholarly journals Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues

PLoS ONE ◽  
2016 ◽  
Vol 11 (3) ◽  
pp. e0152356 ◽  
Author(s):  
Yucheng Zhao ◽  
Jun Luo ◽  
Sheng Xu ◽  
Wei Wang ◽  
Tingting Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Different Tissues ◽  
Selection Of

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

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.

scholarly journals Identification and evaluation of reference genes for quantitative real-time PCR analysis in Polygonum cuspidatum based on transcriptome data

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiaowei Wang ◽  
Zhijun Wu ◽  
Wenqi Bao ◽  
Hongyan Hu ◽  
Mo Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Stress Responses ◽  
Real Time Pcr ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Expression Patterns ◽  
Polygonum Cuspidatum ◽  
Quantitative Real Time Pcr ◽  
Different Tissues

Abstract Background Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data. Results Twelve candidate reference genes, including nine common (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel (SKD1, YLS8, and NDUFA13), were analyzed in different tissues (root, stem, and leaf) without treatment and in leaves under abiotic stresses (salt, ultraviolet [UV], cold, heat, and drought) and hormone stimuli (abscisic acid [ABA], ethylene [ETH], gibberellin [GA3], methyl jasmonate [MeJA], and salicylic acid [SA]). Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes. Conclusions We propose NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species.

scholarly journals Selection of Appropriate Reference Genes for Gene Expression Analysis under Abiotic Stresses in Salix viminalis

2019 ◽  
Vol 20 (17) ◽  
pp. 4210 ◽  
Author(s):  
Valentin Ambroise ◽  
Sylvain Legay ◽  
Gea Guerriero ◽  
Jean-Francois Hausman ◽  
Ann Cuypers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Rank Aggregation ◽  
Salix Viminalis ◽  
Qpcr Data ◽  
Ranking Algorithms ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Selection Of

Salix viminalis is a fast growing willow species with potential as a plant used for biomass feedstock or for phytoremediation. However, few reference genes (RGs) for quantitative real-time polymerase chain reaction (qPCR) are available in S. viminalis, thereby limiting gene expression studies. Here, we investigated the expression stability of 14 candidate reference genes (RGs) across various organs exposed to five abiotic stresses (cold, heat, drought, salt, and poly-metals). Four RGs ranking algorithms, namely geNormPLUS, BestKeeper, NormFinder, and GrayNorm were applied to analyze the qPCR data and the outputs were merged into consensus lists with RankAggreg, a rank aggregation algorithm. In addition, the optimal RG combinations were determined with geNormPLUS and GrayNorm. The genes that were the most stable in the roots were TIP41 and CDC2. In the leaves, TIP41 was the most stable, followed by EF1b and ARI8, depending on the condition tested. Conversely, GAPDH and β-TUB, two genes commonly used for qPCR data normalization were the least stable across all organs. Nevertheless, both geNormPLUS and GrayNorm recommended the use of a combination of genes rather than a single one. These results are valuable for research of transcriptomic responses in different S. viminalis organs.

scholarly journals Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1272
Author(s):  
Judit Tajti ◽  
Magda Pál ◽  
Tibor Janda
Keyword(s):  
Gene Expression ◽  
Avena Sativa ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Nutritional Value ◽  
Tissue Type ◽  
Future Research ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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

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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