Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R.; Mrug, Michal

doi:10.1159/000235993

Cited by 30 publications

(30 citation statements)

References 37 publications

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“…Reactions were performed on a ABI PRISM 7300 Fast real-time PCR system (Applied Biosystems) under the following conditions: 958C for 30 s, 40 cycles of 958C for 5 s, 608C for 31 s to calculate cycle threshold (Ct) values, followed by 958C for 15 s, 608C for 1 min, and then 958C for 15 s to obtain melt curves. The expression of each gene relative to average Ct values of the three housekeeping genes (DCt = Ct gene 2 Ct HKaverage ) was determined using the ABI 7300 system sequence detection software (version 1.4) (Cui et al, 2009;Schuhmacher et al, 2010). To enable direct comparisons with the microarray data, each DCt was added to the averaged GC-RMA-normalized expression values (which are log 2 transformed) of the same three housekeeping genes at the relevant time point from the microarray analysis.…”

Section: Rna Extraction and Transcript Level Analysismentioning

confidence: 99%

Transcriptional Programming and Functional Interactions within the Phytophthora sojae RXLR Effector Repertoire

et al. 2011

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The genome of the soybean pathogen Phytophthora sojae contains nearly 400 genes encoding candidate effector proteins carrying the host cell entry motif RXLR-dEER. Here, we report a broad survey of the transcription, variation, and functions of a large sample of the P. sojae candidate effectors. Forty-five (12%) effector genes showed high levels of polymorphism among P. sojae isolates and significant evidence for positive selection. Of 169 effectors tested, most could suppress programmed cell death triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves. Among the most strongly expressed effectors, one immediate-early class was highly expressed even prior to infection and was further induced 2- to 10-fold following infection. A second early class, including several that triggered cell death, was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. In support of this hypothesis, misexpression of key immediate-early and early effectors severely reduced the virulence of P. sojae transformants.

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Section: Rna Extraction and Transcript Level Analysismentioning

confidence: 99%

Transcriptional Programming and Functional Interactions within the Phytophthora sojae RXLR Effector Repertoire

et al. 2011

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“…Actb is a suitable reference gene for this model based on equivalence testing, a two one-sided t test, 62 of available genomewide expression studies and TaqMan Mouse Endogenous Control Array data for the Cys1 cpk model. 63 The standardized C T values were used to determine significance of studied associations.…”

Section: Gene Expression Analysesmentioning

confidence: 99%

Kidney Injury Accelerates Cystogenesis via Pathways Modulated by Heme Oxygenase and Complement

Zhou¹,

Ouyang²,

Schoeb³

et al. 2012

Journal of the American Society of Nephrology

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AKI accelerates cystogenesis. Because cystogenic mutations induce strong transcriptional responses similar to those seen after AKI, these responses may accelerate the progression of cystic renal disease. Here, we modulated the severity of the AKI-like response in Cys1 cpk/cpk mice, a model that mimics autosomal recessive polycystic kidney disease. Specifically, we induced or inhibited activity of the renoprotective enzyme heme oxygenase (HO) and determined the effects on renal cystogenesis. We found that induction of HO attenuated both renal injury and the rate of cystogenesis, whereas inhibition of HO promoted cystogenesis. HO activity mediated the response of NFkB, which is a hallmark transcriptional feature common to both cystogenesis and AKI. Among the HO-modulated effects we measured, expression of complement component 3 (C3) strongly correlated with cystogenesis, a functionally relevant association as suggested by Cys1 cpk/cpk mice with genetically induced C3 deficiency. Because both C3 deficiency and HO induction reduce cyst number and cyst areas, these two factors define an injury-stimulated cystogenic pathway that may provide therapeutic targets to slow the formation of new renal cysts and the growth of existing cysts.

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“…3). Cui et al [10] recently validated internal RT-PCR controls for use in renal cystic disease (cpk mouse), suggesting Gapdhand Actb genes represent suitable controls. Our results suggest the corresponding protein products, GAPDH and β-actin, are misexpressed in Wpk cystic kidneys and would make poor proteomic controls.…”

Section: Discussionmentioning

confidence: 99%

Differential Expression of Renal Proteins in a Rodent Model of Meckel Syndrome

Mason

Lai

Ringham

et al. 2010

Nephron Exp Nephrol

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Background: Meckel syndrome (MKS) is a fatal autosomal recessive condition with prominent renal cystic pathology. Renal protein misexpression was evaluated in the Wpk rat model of human MKS3 gene disease to identify biomarkers for the staging of renal cystic progression. Methods: Misexpressed proteins were compared between early and late stages of MKS renal cystic disease using proteomic analysis (two-dimensional gel electrophoresis with LC-MS/MS identification) followed by Western blot analysis. Results: A proteomic analysis identified 76 proteins with statistically different, normalized abundance in at least one group. Subsequently, Western blot was used to confirm differential expression in several of these and polycystic kidney disease (PKD)-associated proteins. Galectin-1 and vimentin were identified as overexpressed proteins, which have been previously found in the jck mouse model of nephronophthisis 9. Ciliopathic PKD proteins, polycystins 1 & 2, and fibrocystin were also differentially expressed in Wpk kidney. Conclusion: In the Wpk rat, misexpressed proteins were identified that were also implicated in other forms of cystic disease. Numerous proteins were either over- or underexpressed in late-stage disease. Differences in protein expression may serve as biomarkers of cystic disease and its progression.

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Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues

Cited by 30 publications

References 37 publications

Transcriptional Programming and Functional Interactions within the Phytophthora sojae RXLR Effector Repertoire

Transcriptional Programming and Functional Interactions within the Phytophthora sojae RXLR Effector Repertoire

Kidney Injury Accelerates Cystogenesis via Pathways Modulated by Heme Oxygenase and Complement

Differential Expression of Renal Proteins in a Rodent Model of Meckel Syndrome

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