Identification and Validation of Reference Genes for Gene Expression Analysis in Schima superba

Real-time quantitative PCR (RT-qPCR) 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 fast-growing timber species with strong resistance. However, thus far, reliable reference gene identifications have not been reported in S. superba. In this study, 19 candidate reference genes were selected and evaluated for their expression stability in different tissues of S. superba. 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 show that SsuACT was the most stable reference gene and that SsuACT + SsuRIB was the best reference gene combination for different tissues. Finally, the stable and less stable reference genes were verified using SsuSND1 expression in different tissues. To our knowledge, this is the first report to verify appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate the future elucidation of gene regulations in this species and useful references for relative species.

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Identification and validation of appropriate reference genes for gene expression analysis in Schima superba

10.21203/rs.3.rs-361995/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 ◽

Real Time Quantitative Pcr ◽

Stability Ranking ◽

Different Tissues

Abstract Background: Real-time quantitative PCR (RT-qPCR) 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, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

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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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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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Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽

10.7717/peerj.3260 ◽

2017 ◽

Vol 5 ◽

pp. e3260 ◽

Cited By ~ 4

Author(s):

Kai Wang ◽

Yi Niu ◽

Qijun Wang ◽

Haili Liu ◽

Yi Jin ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Reference Genes ◽

Dynamic Range ◽

Expression Profiles ◽

Mrna Levels ◽

Expression Stability ◽

Degenerate Primers ◽

Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

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Expression Stability of Internal Reference Gene in Response to Trichoderma polysporum Infection in Avena fatua L.

10.21203/rs.3.rs-507940/v1 ◽

2021 ◽

Author(s):

Haixia Zhu ◽

Yongqiang Ma ◽

Liang Cheng

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Avena Fatua ◽

Expression Stability ◽

Internal Reference ◽

Qpcr Analysis ◽

Suitable Combination

Abstract In order to construct a RT-qPCR system suitable for response of Avena fatua L. to Trichoderma polysporum , and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on 8 candidate internal reference genes ( 18S , 28S , TUA , UBC , ACT , GAPDH , TBP and EF-1 ) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP , 18S and UBC were the most stable internal reference genes, TBP and TUA , TBP and GAPDH , 18S and TBP , UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as the internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua .

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Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

BioMed Research International ◽

10.1155/2016/8367063 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 9

Author(s):

Zhaoping Yan ◽

Jinhang Gao ◽

Xiuhe Lv ◽

Wenjuan Yang ◽

Shilei Wen ◽

...

Keyword(s):

Gene Expression ◽

Acute Pancreatitis ◽

Reference Gene ◽

Reference Genes ◽

Suitable Reference Gene ◽

Expression Stability ◽

Rt Pcr ◽

Comprehensive Method ◽

Suitable Combination ◽

Suitable Reference

The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α> 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis.

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Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca

F1000Research ◽

10.12688/f1000research.2-37.v1 ◽

2013 ◽

Vol 2 ◽

pp. 37 ◽

Cited By ~ 2

Author(s):

Teresa Docimo ◽

Gregor W Schmidt ◽

Katrin Luck ◽

Sven K Delaney ◽

John C D'Auria

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Tropane Alkaloid ◽

Expression Stability ◽

Internal Reference ◽

Real Time Quantitative Pcr ◽

Gene Expression Studies ◽

The Stability ◽

High Level ◽

Pharmacologically Active

Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis.Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca (Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

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Evaluation And Validation of Reliable Reference Genes For Quantitative Real-Time PCR Analysis of The Gene Expression In Macadamia Integrifolia

10.21203/rs.3.rs-645521/v1 ◽

2021 ◽

Author(s):

Qian Yang ◽

Ziping Yang ◽

Yali Zhou ◽

Hui Zeng ◽

Minghong Zou ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Geometric Mean ◽

Elongation Factor ◽

Stable Gene ◽

Expression Stability ◽

Tissue Samples ◽

Qrt Pcr ◽

Macadamia Integrifolia ◽

Elongation Factor 1

Abstract Background: Macadamia integrifolia, a new economically important crop, the kernel oil is rich in bioactive compound and monounsaturated fatty acid. Gene expression analysis of qRT-PCR is beneficial to understand the complex regulatory networks of macadamia.Results: In this study, the expression stability of 11 traditional housekeeping genes including α-tubulin (TUBa), β–tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosome RNA (18S), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β- elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP) and actin (ACT) were accessed by qRT-PCR in macadamia seedlings under different experimental conditions and tissues. The expression stability of the 11 reference genes was evaluated by the online tool RefFinder, which include ΔCt, geNorm, NormFinder, BestKeeper four commonly software, and then determinated a comprehensive expression stability ranking by integrating above four ranking results based on the geometric mean. Our results show that ACT was the best stable genes for all samples, cold stress, NaCl sress, PEG stress, ABA treatment, MeJA treatment, stem and leaf tissue samples; EF1b is the most stable gene in GA treatment and heat stress samples; UBC and CYP were respectively ranked top in ethylene treatment and root tissue samples. Finally, the reliability of these results was further validated with a target gene SAD by qRT-PCR. Conclusions: In summary, this study evaluated and validated the suitable reference genes for qRT-PCR under different experiment treatment and tissues, and will be useful for further gene expression studies on the molecular mechanisms in Macadamia.

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Evaluation of Housekeeping Gene Expression Stability in Carnation (Dianthus caryophyllus)

10.21203/rs.2.24535/v1 ◽

2020 ◽

Author(s):

Huolin Luo ◽

Wenjing Yu ◽

Yuan Tao ◽

Jonathan Hrovat ◽

Ahui Xue ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Dianthus Caryophyllus ◽

Housekeeping Gene ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Suitable Reference Gene ◽

Expression Stability ◽

Experimental Conditions

Abstract Background: The real-time quantitative reverse transcription PCR (RT-qPCR) is widely used for gene expression analysis, owing to its advantages of high specificity, sensitivity and repeatability. A suitable reference gene is an absolute prerequisite for accurate normalization, nevertheless, the frequently-used reference gene was reported unstable under different experimental conditions and causes failure to correctly analyze the expression of the interested gene. Therefore, it is vital to systematically evaluate the expression stability of these candidate reference genes before performing RT-qPCR. Results: In this study, two computational statistical methods were used, including geNorm and NormFinder, in order to determine the expression stability of 12 frequently-used reference genes in Dianthus caryophyllus across different experimental conditions. The results show that the expression stability of candidate genes varies greatly in different sample pools, which again proves the instability of these common housekeeping gene expressions. In general, the expression of UBQ10 (ubiquitin10), EF1a (elongation factor 1A) and TIP41 (TIP41-like family protein) were relatively stable under different experimental conditions, while the expression stability of 18S (18S Ribosome RNA), TIF5A (translation initiation factor 5A) and PP2A (protein phosphatase 2A) were relatively poor. Conclusion: EF1α, TIP41 and UBQ10 were considered the most appropriate reference genes when all samples were put together, while UBQ10 was most stable in exogenous hormone treatments. TUB and UBQ10 can be used as reliable internal control genes under stress, while CYP and TUA can act as reliable internal controls in different tissues. This is the first systematic study of selection of reference genes in Carnation, and will benefit future expression studies in this crop.

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Evaluation of Candidate Reference Genes for Quantitative Gene Expression Studies in Tree Peony

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.141.2.99 ◽

2016 ◽

Vol 141 (2) ◽

pp. 99-111 ◽

Cited By ~ 4

Author(s):

Lin Zhou ◽

Qianqian Shi ◽

Yan Wang ◽

Kui Li ◽

Baoqiang Zheng ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Developmental Stages ◽

Expression Patterns ◽

Ornamental Plants ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Expression Stability ◽

Tree Peony ◽

Different Tissues

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) is a sensitive and widely used technique for gene expression analysis that depends on stability of the reference genes used for data normalization. Tree peony (Paeonia suffruticosa), known as one of the most famous traditional ornamental plants in China, is very popular in both domestic and international markets for its showy and colorful flowers. To date, no systematic studies on reference genes have been performed in tree peony with different flower colors. In this study, we evaluated the expression stability of 12 candidate reference genes in different tissues and five flower developmental stages of tree peony with six different colors by three algorithms: geNorm, NormFinder, and BestKeeper. The results showed that protein phosphatase 2A (PP2A), ubiquitin protein ligase (UPL), and ubiquitin (UBQ) were the most stable genes across all samples. Helicase, alpha-tubulin (TUA), and eukaryotic translation initiation factor 5A (EIF5A) also exhibited high expression stability in different tissues, in samples with different colors, and at different flower developmental stages. According to the geNorm analysis, the combination of two most stable reference genes was optimal for normalization in all tested sample sets in this study. To further validate the suitability of the reference genes identified in this study, the expression patterns of two putative homologs of chalcone synthase gene (PsCHS1) and chalcone isomerase gene (PsCHI1) were studied at different developmental stages of white flowers. The results provide information for transcriptional analyses in future studies of gene expression on tree peony flower development and pigmentation.

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