Establishment of reference (housekeeping) genes via quantitative real-time PCR for investigation of the genomic basis of abiotic stress resistance in Psammochloa villosa (Poaceae)

Liu, Yu Ping; Zhang, Yu; Liu, Feng; Liu, Tao; Chen, Jin‐Yuan; Fu, Gui Hong; Zheng, Chang Yuan; Su, Dan; Wang, Ya Nan; Zhou, Hua; Su, Xu; Aj, Harris; Wang, Xiu Mei

doi:10.1016/j.jplph.2021.153575

Cited by 6 publications

(8 citation statements)

References 71 publications

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“…These candidate reference genes included 8 traditional reference genes ( ACT7 , ACT11 , TUB , ERF3A , TBCB , GAPDH , EF-1α and His ) and two novel genes, DIM1 and R3H. Primer Premier 5.0 software [ 57 ] was used to design the candidate reference gene primers. The parameters were set as follows: melting temperature (Tm), 55–65℃; GC content, 40–60%; primer length, 17–26 bp; and amplification product size, 150–300 bp [ 21 , 58 ].…”

Section: Methodsmentioning

confidence: 99%

Selection and validation of appropriate reference genes for RT–qPCR analysis of Nitraria sibirica under various abiotic stresses

Yang

Zhu

et al. 2022

BMC Plant Biol

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Background Nitraria sibirica Pall. is a halophytic shrub with strong environmental adaptability that can survive in extremely saline-alkali and drought-impacted environments. Gene expression analysis aids in the exploration of the molecular mechanisms of plant responses to abiotic stresses. RT–qPCR is the most common technique for studying gene expression. Stable reference genes are a prerequisite for obtaining accurate target gene expression results in RT–qPCR analysis. Results In this study, a total of 10 candidate reference genes were selected from the transcriptome of N. sibirica, and their expression stability in leaves and roots under different treatment conditions (salt, alkali, drought, cold, heat and ABA) was evaluated with the geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the tissue and experimental conditions tested. ACT7 combined with R3H, GAPDH, TUB or His were the most stable reference genes in the salt- or alkali-treated leaves, salt-treated roots and drought-treated roots, respectively; R3H and GAPDH were the most suitable combination for drought-treated leaves, heat-treated root samples and ABA-treated leaves; DIM1 and His maintained stable expression in roots under alkali stress; and TUB combined with R3H was stable in ABA-treated roots. TBCB and GAPDH exhibited stable expression in heat-treated leaves; TBCB, R3H, and ERF3A were stable in cold-treated leaves; and the three most stable reference genes for cold-treated roots were TBCB, ACT11 and DIM1. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of the NsP5CS gene under the six treatment conditions. Conclusion This study provides a theoretical reference for N. sibirica gene expression standardization and quantification under various abiotic stress conditions and will help to reveal the molecular mechanisms that confer stress tolerance to N. sibirica.

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Section: Methodsmentioning

confidence: 99%

Selection and validation of appropriate reference genes for RT–qPCR analysis of Nitraria sibirica under various abiotic stresses

Yang

Zhu

et al. 2022

BMC Plant Biol

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“…For example, the stable expression of GAPDH in grape berries is the worst in wheat ( Triticum aestivum ) [ 11 , 12 ]. In the stems of Psamochloa villosa , the ACT gene is the most suitable internal reference gene for gene expression research under drought conditions, while tubulin alfa chain ( TUA) is the most stable and the best internal reference gene under cold conditions [ 13 ]. Therefore, screening internal reference genes in accordance with different species, tissues, and experimental conditions is necessary to ensure the accuracy of RT-qPCR results [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata

Wang

Qian

Geng

et al. 2023

Genes

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Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai–Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

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“…The quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) technique has been widely used for transcript quantification due to its high accuracy, sensitivity, specificity, low cost, and high-throughput characteristics [1][2][3][4][5][6][7]. Gene expression can be significantly influenced by a series of steps in an experimental procedure, such as the initial material amount, the quality of mRNA templates, reverse-transcription efficiency, and cDNA quality [1,[8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…However, numerous studies have shown that the transcript levels of these housekeeping genes vary considerably in response to variable experimental conditions, different tissue types, and/or across taxa [6,7,14,[18][19][20][21]; thus, none of the commonly developed genes reported so far can act as a universal reference. Hence, it is necessary to select a set of appropriate reference genes for the specific set of chosen experimental conditions and tissue types [13,[22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

“…Benefitting from high-throughput sequencing technology, growing plant transcriptome datasets have provided potential resources for the identification of appropriate reference genes [6,7,[31][32][33]. In this study, 14 candidate reference genes (TUA4, TUB6, ROC3, CYP2, GAPC2, YLS8, PTB1, TIP41, EXP1, EXP2, ACT2, PP2AA3, EF1-α, and UBC9) were assembled from the nasturtium seed transcriptome database (Table 1).…”

Section: Introductionmentioning

confidence: 99%

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Selection and Validation of Reference Genes for qRT-PCR Analysis of Gene Expression in Tropaeolum majus (Nasturtium)

Tang,

Zhou,

Liu

et al. 2023

Horticulturae

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Tropaeolum majus (nasturtium) is an important ornamental and medicinal plant due to its colorful flowers, shield-shaped leaves, and richness in mineral elements and bioactive compounds. However, the key genes related to these important biological traits, as well as their expression patterns and functions, remain obscure. In this study, to choose appropriate reference genes for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, we screened 14 candidate genes from the transcriptome of T. majus and evaluated their expression stability. Through evaluation with four commonly used algorithms (geNorm, NormFinder, BestKeeper, and RefFinder), EXP1, EXP2, and TUB6 were found to be the most stably expressed genes among different organs, while EXP1 combined with CYP2 was identified as the optimal reference gene combination for seeds at different development stages. For all the tested samples, EXP1, EXP2, CYP2, and ACT2 were the most suitable reference genes. Moreover, the target gene KCS11 involved in very-long-chain fatty acid biosynthesis was employed to confirm the most and least stable reference genes in different organs, seeds at different development stages, and all the tested samples. The expression profiles of KCS11 were similar, with minor differences based on the analysis of different stable reference genes (either alone or in combination), while the expression profiles were diverse and the relative expression level was overestimated when using the least stable ones. These results suggest that the appropriate selection of reference genes is critical for the normalization of gene expression. Furthermore, the reference genes screened in this study will greatly improve the accuracy of the qRT-PCR quantification of candidate genes involved in the many biological characteristics of nasturtium.

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Establishment of reference (housekeeping) genes via quantitative real-time PCR for investigation of the genomic basis of abiotic stress resistance in Psammochloa villosa (Poaceae)

Cited by 6 publications

References 71 publications

Selection and validation of appropriate reference genes for RT–qPCR analysis of Nitraria sibirica under various abiotic stresses

Selection and validation of appropriate reference genes for RT–qPCR analysis of Nitraria sibirica under various abiotic stresses

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata

Selection and Validation of Reference Genes for qRT-PCR Analysis of Gene Expression in Tropaeolum majus (Nasturtium)

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