Simple regression for correcting ΔCt bias in RT-qPCR low-density array data normalization

Cui, Xiangqin; Yu, Shaohua; Tamhane, Ashutosh; Causey, Zenoria; Steg, Adam D.; Danila, Maria I.; Reynolds, Richard J.; Wang, Jinyi; Wanzeck, Keith; Tang, Qi; Ledbetter, Stephanie; Redden, David T.; Johnson, Martin R.; Bridges, S. Louis

doi:10.1186/s12864-015-1274-1

Cited by 19 publications

(17 citation statements)

References 32 publications

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“…com/pebiodocs/00113186.pdf). This was validated by linear regression with Gapdh as the reference gene (Cui et al 2015) with adjustment for sample group (included as an additional independent variable). The expression of the remaining genes is presented as 2…”

Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles

2015

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Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67 or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67 and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation. Reproduction (2015) 150 11-20

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Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles

2015

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“…Expression values of target genes are traditionally corrected to the expression of the control gene using a ΔCt approach, but this assumes that the expression of the control gene is kept constant across conditions. Since our experiment is likely to influence overall homeostasis, and therefore impact control gene expression, we obtained normalized Ct values using the method of Cui et al . (2015): normalized value = target_Ct value – ( b x control_Ct value) where b is the regression coefficient of the linear regression of mean CYP2J19 Ct values on mean β-actin Ct values.…”

Section: Methodsmentioning

confidence: 99%

A mitochondria-targeted antioxidant and a thyroid hormone affect carotenoid ketolase gene expression and bill redness in zebra finches

Cantarero

Andrade

Carneiro

et al. 2020

Preprint

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Conspicuous ornaments in animals can evolve to reveal individual quality when their production/maintenance costs make them reliable as signals or if their expression level is intrinsically linked to quality by some unfalsifiable mechanism (quality indices). The latter has been mostly associated with traits constrained by body size. However, red ketocarotenoid-based coloured ornaments may also have evolved as quality indices because their production could be closely linked to individual metabolism and, particularly, to the cell respiration at the inner mitochondrial membrane (IMM). This mechanism would supposedly not depend on resource (yellow carotenoids) availability, thus discarding allocation trade-offs. A gene coding for a ketolase enzyme (CYP2J19) responsible for converting dietary yellow carotenoids to red ketocarotenoids has recently been described in birds. It is not known, however, if this ketolase is involved in mitochondrial metabolism and if its expression level and activity is resource independent. Here, we manipulated the metabolism of captive male zebra finches by an antioxidant designed to penetrate the IMM (mitoTEMPO) and a thyroid hormone (triiodothyronine; T3) with known hypermetabolic effects. The expression levels of a ketocarotenoid-based ornament (bill redness) and CYP2J19 were measured. MitoTEMPO downregulated CYP2J19 expression, supporting the mitochondrial involvement in ketolase function. T3 also reduced CYP2J19 expression, but at an intermediate dosage, this effect being buffered by mitoTEMPO. Bill redness seemed to show a similar interacting effect. Nevertheless, this faded when CYP2J19 expression level was controlled for as a covariate. We argue that the well-known mitoTEMPO effect in reducing mitochondrial reactive oxygen species (ROS) production (particularly superoxide) could have interfered on redox signalling mechanisms controlling ketolase transcription. High T3 levels, contrarily, can lead to high ROS production but also trigger compensatory mechanisms, which may explain the U-shaped effect with dosage on CYP2J19 expression levels. Bill CYP2J19 expression values were also positively correlated to redness and circulating substrate carotenoid levels. Nonetheless, treatment effects did not change when controlling for blood carotenoid concentration, suggesting that resource-availability dependence was irrelevant. Finally, our findings reveal a role for thyroid hormones in the expression of carotenoid-based ornaments that has virtually been ignored until now.

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“…All PCRs were performed in triplicate, and the specificity of the reactions was detected by melting-curve analysis at the dissociation stage. Each target gene was comparatively quantified based on cycle threshold (CT) normalized to GhGAPDH, using the DDCT method (Cui et al, 2015).…”

Section: Rna Extraction and Real-time Quantitative Polymerase Chain Rmentioning

confidence: 99%

Methylation-sensitive amplified polymorphism analysis of Verticillium wilt-stressed cotton (Gossypium)

et al. 2016

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In this study, a methylation-sensitive amplification polymorphism analysis system was used to analyze DNA methylation level in three cotton accessions. Two disease-sensitive near-isogenic lines, PD94042 and IL41, and one disease-resistant Gossypium mustelinum accession were exposed to Verticillium wilt, to investigate molecular disease resistance mechanisms in cotton. We observed multiple different DNA methylation types across the three accessions following Verticillium wilt exposure. These included hypomethylation, hypermethylation, and other patterns. In general, the global DNA methylation level was significantly increased in the disease-resistant accession G. mustelinum following disease exposure. In contrast, there was no significant difference in the disease-sensitive accession PD94042, and a significant decrease was observed in IL41. Our results suggest that disease-resistant cotton might employ a mechanism to increase methylation level in response to disease stress. The differing methylation patterns, together with the increase in global DNA methylation level, might play important roles in tolerance to Verticillium wilt in cotton. Through cloning and analysis of differently methylated DNA sequences, we were also able to identify several genes that may contribute to disease resistance in cotton. Our results revealed the effect of DNA methylation on cotton disease resistance, and also identified genes that played important roles, which may shed light on the future cotton disease-resistant molecular breeding.

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Simple regression for correcting ΔCt bias in RT-qPCR low-density array data normalization

Cited by 19 publications

References 32 publications

Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles

Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles

A mitochondria-targeted antioxidant and a thyroid hormone affect carotenoid ketolase gene expression and bill redness in zebra finches

Methylation-sensitive amplified polymorphism analysis of Verticillium wilt-stressed cotton (Gossypium)

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