Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination

Mosley, Yung Yi C.; HogenEsch, Harm

doi:10.1186/s13104-017-3005-y

Cited by 10 publications

(9 citation statements)

References 33 publications

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“…Top 15 candidate reference genes with the lowest CV values are all annotated proteins, and most of them have been widely studied in model organisms. For example, UBC has been identified as a reliable reference gene in some organisms [26][27][28]. By GO enrichment analysis of all candidate reference genes, as expected, candidates are highly enriched in biological process associated with metabolic process, cellular metabolic process, and protein metabolic process, suggesting they may play an essential part in maintenance of basal cellular functions, as described in other organisms of human, mouse, maize, and scallop [29][30][31].…”

Section: Stability Analysis Between Candidates and Commonly Used Refesupporting

confidence: 61%

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Jin¹,

Song²,

Wang³

et al. 2020

Preprint

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Background: The quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used technique that relies on the reference gene for gene expression normalization. Selecting a suitable reference gene is a crucial step to obtain an accurate result in qRT-PCR. However, most previous studies of fishes adopted reference genes that were commonly used in mammals without validation. Results: In this study, we utilized 89 transcriptome datasets covering early developmental stages and adult tissues, and carried out transcriptome-wide identification and validation of reference genes in Sebastes schlegelii. Finally, 121 candidate reference genes were identified based on four criteria. Eight candidates (METAP2, BTF3L4, EIF5A1, TCTP, UBC, PAIRB, RAB10, and DLD) and four commonly used reference genes (TUBA, ACTB, GAPDH, RPL17) in mammals were selected for validation via qRT- PCR and four statistical methods (delta-Ct, BestKeeper, geNorm, and NormFinder). The results revealed that the candidate reference genes we recommended are more stable than traditionally used ones. Conclusions: This is the first study to conduct transcriptome-wide identification and validation of reference genes for quantitative RT-PCR in the black rockfish, and lay an important foundation for gene expression analysis in teleost.

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Section: Stability Analysis Between Candidates and Commonly Used Refesupporting

confidence: 61%

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Jin¹,

Song²,

Wang³

et al. 2020

Preprint

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“…Therefore, when such a great variation is observed on the genes ranked by different tools, there is no recommendation of the best method to select the genes, as well as there is no standard score or threshold indicating good or bad stability. Mosley et al (2017) [ 37 ], after analyzing 5 tools (BestKeeper, geNorm, NormFinder, DeltaCt and RefFinder), concluded that the geNorm seems to be the best tool for choosing the most reliable genes.…”

Section: Discussionmentioning

confidence: 99%

Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs

et al. 2018

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The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue.

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“…Although there is a great variation in the classification of genes by different software, there is no recommendation for the best method of gene selection, nor a pattern that indicates good or poor stability [46]. After the analysis with BestKeeper, geNorm and NormFinder, the geNorm seemed to be the most suitable software to choose the most reliable genes, because its results were more similar to those found with RankAggreg.…”

Section: Plos Onementioning

confidence: 96%

Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

et al. 2020

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The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies.

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Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination

Cited by 10 publications

References 33 publications

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs

Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

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