Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Mo, Fei; Zhao, Jie; Liu, Na; Cao, Lihua; Jiang, Shanxiang

doi:10.1590/s1415-47572014000400005

Cited by 20 publications

(10 citation statements)

References 32 publications

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“…Normfinder algorithm considers variations unrestricted to different samples, but includes differences between and within experimental groups. Such variations are very important factors to be considered in reference gene validation, as demonstrated in the present study, where significant difference (P < 0.05) between resistant and susceptible groups was observed for GAPDH Cq values in the abomasal pyloric region, which corroborated the findings of other studies [20,32,44]. Another advantage of this program is the suggestion of combinations of reference genes instead using only one.…”

Section: Discussionsupporting

confidence: 91%

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

et al. 2018

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Due to the great economic impact of Haemonchus contortus on sheep farming, there is an increasing number of studies addressing host resistance against this nematode, including identification of directly related immune mechanisms. In this context, relative gene expression by RT-qPCR have been largely used, due to its rapidity, high sensitivity, specificity, and reproducibility. Although, appropriate reference gene selection is crucial for accurate interpretation of results. In this study, five reference genes (GAPDH, G6PDH, YWHAZ, ACTB, and B2M) were tested for expression stability in abomasum (fundic and pyloric regions) and abomasal lymph nodes of Morada Nova sheep classified as resistant (n = 5) or susceptible (n = 5) to H. contortus infection in a flock of 151 animals. GAPDH combined with YWHAZ were selected as reference genes for abomasal fundic region and abomasal lymph nodes, whereas YWHAZ was the most stable gene for abomasal pyloric region. These genes presented the lowest intra- and inter-group variations and, consequently, highest stability. In contrast, expression of G6PDH was the least stable in all tissues. The impact of reference gene selection was demonstrated by relative quantification of a pro-inflammatory cytokine (TNFα) in abomasal fundic region. Significant differences in TNFα expression levels between resistant and susceptible groups were only observed when the most stable genes (GAPDH combined with YWHAZ) or GAPDH were used as reference genes, whereas no significant differences were observed when other tested reference genes were used. It was demonstrated that normalization of expression data using inappropriate reference genes may significantly influence interpretation results.

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Section: Discussionsupporting

confidence: 91%

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

et al. 2018

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“…To the best of our knowledge, there is no gold standard for the optimal number of the candidate reference genes required for the expression stability study. In general, six to twelve candidate genes are commonly included for the analysis 59 – 76 . The supply of cDNA samples and the lab resources dedicated for the characterization should also be considered to make the final decision on the number of candidate genes used for the analysis.…”

Section: Discussionmentioning

confidence: 99%

Identification of reference genes for qRT-PCR in granulosa cells of healthy women and polycystic ovarian syndrome patients

Zhao

Lü

et al. 2017

Sci Rep

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Comparative gene expression analysis by qRT-PCR is commonly used to detect differentially expressed genes in studies of PCOS pathology. Impaired GC function is strongly associated with PCOS pathogenesis, and a growing body of studies has been dedicated to identifying differentially expressed genes in GCs in PCOS patients and healthy women by qRT-PCR. It is necessary to validate the expression stability of the selected reference genes across the tested samples for target gene expression normalization. We examined the variability and stability of expression of the 15 commonly used reference genes in GCs from 44 PCOS patients and 45 healthy women using the GeNorm, BestKeeper, and NormFinder statistical algorithms. We combined the rankings of the three programs to produce a final ranking based on the geometric means of their stability scores. We found that HPRT1, RPLP0, and HMBS out of 15 examined commonly used reference genes are stably expressed in GCs in both controls and PCOS patients and can be used for normalization in gene expression profiling by qRT-PCR. Future gene-expression studies should consider using these reference genes in GCs in PCOS patients for more accurate quantitation of target gene expression and data interpretation.

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“…The eef1a1 gene encodes an isoform of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. It is a commonly used reference gene in X. laevis , 17 and in other animal species, 30 plants 31 and cell lines. 32 Although it is commonly used, eef1a1 should not be generalised as the perfect reference gene.…”

Section: Discussionmentioning

confidence: 99%

Reference Gene Validation for Quantitative Real-time PCR Studies in Amphibian Kidney-derived A6 Epithelial Cells

2019

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Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.

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Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Cited by 20 publications

References 32 publications

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

Identification of reference genes for qRT-PCR in granulosa cells of healthy women and polycystic ovarian syndrome patients

Reference Gene Validation for Quantitative Real-time PCR Studies in Amphibian Kidney-derived A6 Epithelial Cells

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