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

Wei, Tong-Lu; Wang, H.; Liu, H.‐N.; Yu, Yi‐He; Jiang, Jianfu; Guo, Da‐Long

doi:10.1111/ajgw.12483

Cited by 10 publications

(4 citation statements)

References 55 publications

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“…Many studies on reference gene selection were reported in kiwifruit, with selected genes including TUB and ACTB combination and ACT, GAPDH, and UBQ combination [45,46,50]. In fig stems, 18s rRNA emerged as the preferred reference gene [47], In grape, RRM1 and EF-1α in combination are appropriate as internal reference genes [51]. For grape branch and leaf development processes, Ren et al [52] identified GAPDH, UBQ-1, and EF-1α1 as suitable reference genes.…”

Section: Selection Of Internal Reference Genes In Vegetative Organsmentioning

confidence: 99%

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Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review

Peng,

Ali Sabir,

et al. 2024

IJMS

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Real-time quantitative polymerase chain reaction (qRT-PCR) has been widely used in gene expression analyses due to its advantages of sensitivity, accuracy and high throughput. The stability of internal reference genes has progressively emerged as a major factor affecting the precision of qRT-PCR results. However, the stability of the expression of the reference genes needs to be determined further in different cells or organs, physiological and experimental conditions. Methods for evaluating these candidate internal reference genes have also evolved from simple single software evaluation to more reliable and accurate internal reference gene evaluation by combining different software tools in a comprehensive analysis. This study intends to provide a definitive reference for upcoming research that will be conducted on fruit trees. The primary focus of this review is to summarize the research progress in recent years regarding the selection and stability analysis of candidate reference genes for different fruit trees.

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Section: Selection Of Internal Reference Genes In Vegetative Organsmentioning

confidence: 99%

“…UQB was also deemed suitable for the leaves of litchi [58] and apple [59]. For grape leaves, GAPDH, UBQ-1, EF-1α1, RRM1, and EF-1α combinations were found to be appropriate internal reference genes [51,52].…”

Section: Selection Of Internal Reference Genes In Vegetative Organsmentioning

confidence: 99%

Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review

Peng,

Ali Sabir,

et al. 2024

IJMS

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“…It is generally the housekeeping gene that maintains the basic life activities of cells, such as Actin, 18s Ribosome RNA, Tublin and Ubiquitin, etc. [19,20]. However, many studies have proved that the transcription levels of the housekeeping genes may change with different species, tissues, and organs [21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera

Chen,

Wang,

Hao

et al. 2023

IJMS

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Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO4 treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

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“…Two independent studies have used microarray data to search for the most stable novel reference genes of wine grape cultivars under biotic stress (i.e., infection with Plasmopara viticola and Botrytis cinerea) [64], or abiotic stress (water and heat stress) [65]. Two other studies have used RNA-Seq data to identify novel reference genes in berries of table grapes across different phenological stages and under abiotic stress [66,67]. However, no studies have used genome-wide screening to identify reference genes with stable expression across viral infection, tissue types and phenological stages.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

2021

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Background Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. Results We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. Conclusions We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.

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

Cited by 10 publications

References 55 publications

Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review

Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

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