Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

Hu, Yu‐Ning; Fu, Hongtuo; Qiao, Hui; Sun, Shengming; Zhang, Wenyi; Jin, Shubo; Jiang, Sufei; Gong, Yongsheng; Xiong, Yiwei; Wu, Yan

doi:10.3390/ijms19082258

Cited by 75 publications

(58 citation statements)

References 41 publications

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“…6). Considered to be an integrative statistical program, RefFinder has been widely applied to evaluate the overall stability of reference gene expression and determine appropriate reference genes for diverse plant species [31, 48]. Based on the comprehensive RefFinder analysis, we have summarized and demonstrated the ordering of reference gene stability under various treatments and tissues (Fig.…”

Section: Discussionmentioning

confidence: 99%

Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum

Zhang

Jiang

et al. 2018

Plant Methods

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BackgroundReal-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley (Hordeum brevisubulatum (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and extremely wide adaptation. However, suitable references gene have not been documented for standardization of gene expression in wild barley under abiotic stress.ResultsHere we report the first systematic and comprehensive analysis of reference genes for quantitative real-time PCR standardization in wild barley. We selected 11 genes, including ACT (Actin), ADP (ADP-ribosylation factor 1), CYP2 (Cyclophilin 2), EF-1α (Elongation factor 1-alpha), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), HSP90 (Heat shock protein 90), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), UBI (Ubiquitin), 18SrRNA-1 (guanine1575-N7-methyltransferase) and 18SrRNA-3 (adenine1779-N6-dimethyltransferase) from a wild barley transcriptome database and analyzed their expression stabilities in shoots and roots of wild barley seedling under various stress conditions using comparative ΔCt, BestKeeper, Normfinder and geNorm software. The results demonstrated that ADP was the most suitable reference gene in salt stress while UBI showed peak stability under mannitol and ABA stress; EF-1α was the most appropriate reference gene for PEG, GA3, ethylene and heat stress; 18SrRNA-3 was the best choice for cold stress; and TUBα was the first stable gene across different tissues.ConclusionsOur main contribution was to identify reference genes with suitable and stable expression in wild barley under various stress conditions and in different tissues to provide a useful resource for future studies. The results demonstrate the importance of transcriptome data as a useful resource for the screening of candidate reference genes and highlight the need for specific reference genes for specific conditions. Furthermore, these findings will provide valuable information for wild barley and relative species for future research.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0379-3) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum

Zhang

Jiang

et al. 2018

Plant Methods

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“…Beta actin was constantly smooth expressed in different developmental stages of prawns. Bestkeeper analysis and similar methods were performed for expression levels of beta actin [43]. Differences in expression levels were considered significant at p < 0.05.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense

Wang

Jin

et al. 2019

IJMS

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The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.

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“…The qRT-PCR technique is one of the most valuable and reliable research tools used to quantify the expression of a target gene under different experimental conditions. Proper use of NFs is necessary to get a reliable estimate of gene expression in different types of breast cancer tissues and cells to avoid detecting variations that are not cancer-speci c [43][44][45]. Therefore, the selection of the appropriate RGs for breast cancer research is important when using qRT-PCR to quantify gene expression.…”

Section: Discussionmentioning

confidence: 99%

Selecting optimal reference genes for breast cancer research

Song¹,

Huang²,

Wu³

et al. 2020

Preprint

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Background Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes (RGs) is critical for normalizing and evaluating changes in the expression of target genes. However, uniform and reliable RGs for breast cancer research have not been identified, limiting the value of target gene expression studies. Here, we provide a novel approach for mining RGs by using the RNA-seq dataset to identify reliable and accurate RGs that can be applied to different types of breast cancer tissues and cell lines. Methods First, we compiled the transcriptome profiling data from the TCGA database involving 1217 samples to identify novel RGs and then ten genes (SF1, TARDBP, THRAP3, QRICH1, TRA2B, SRSF3, YY1, DNAJC8, RNF10, and RHOA) with relatively stable expression levels were chosen as novel candidate RGs. Additionally, six conventional RGs (ACTB, TUBA1A, RPL13A, B2M, GAPDH, and GUSB) were also selected. To determine and validate the optimal RGs we performed qRT-PCR experiments on 87 samples from 5 types of surgically excised breast tumor specimens including HR+HER2-, HR+HER2+, HR-HER2-, HR-HER2+, breast cancer after neoadjuvant chemotherapy (NAC) and their matched para-carcinoma tissues, furthermore, we also included a benign breast tumor sample. Six biological replicates were included for each tissue. Moreover, we assessed 7 breast cancer cell lines (MCF-10A, MCF-7, T-47D, MDA-MB-231, MDA-MB-468, as well as MDA-MB-231 with either CNR2 knockdown or overexpression; 3 biological replicates for each line). Five statistical algorithms (geNorm, NormFinder, ΔCt method, BestKeeper, and ComprFinder) were used to assess the stability of expression of each RG across all breast cancer tissues and cell lines. Results Our results show that RG combinations SF1+TRA2B+THRAP3 and THRAP3+RHOA+QRICH1 showed stable expression in breast cancer tissues and cell lines, respectively, and that these two combinations displayed good interchangeability. Therefore, we propose that the above two combinations are optimal triplet RGs for breast cancer research. Conclusions In summary, we identified novel and reliable RG combinations for breast cancer research based on a public RNA-seq dataset which lays a solid foundation for accurate normalization of qRT-PCR results across different breast cancer tissues and cells.

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Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

Cited by 75 publications

References 41 publications

Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum

Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense

Selecting optimal reference genes for breast cancer research

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