Quantitative Reverse Transcription–Polymerase Chain Reaction to Study mRNA Decay: Comparison of Endpoint and Real-Time Methods

Schmittgen, Thomas D.; Zakrajsek, Brian A.; Mills, Alan; Gorn, V. V.; Singer, Michael J.; Reed, Michael W.

doi:10.1006/abio.2000.4753

Cited by 922 publications

(625 citation statements)

References 18 publications

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“…PCR reactions were carried out in triplicate using 100 ng cDNA from both patient and control samples. Levels of mRNA were relatively quantified by evaluating Ct values using the comparative Ct (ΔCt) method [33].…”

Section: Glycosaminoglycans Determinationmentioning

confidence: 99%

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

et al. 2015

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Aminoglycoside antibiotics, such as gentamicin, may induce premature termination codon (PTC) readthrough and elude the nonsense-mediated mRNA decay mechanism. Because PTCs are frequently involved in lysosomal diseases, readthrough compounds may be useful as potential therapeutic agents. The aim of our study was to identify patients responsive to gentamicin treatment in order to be used as positive controls to further screen for other PTC readthrough compounds. With this aim, fibroblasts from 11 patients affected by 6 different lysosomal diseases carrying PTCs were treated with gentamicin. Treatment response was evaluated by measuring enzymatic activity, abnormal metabolite accumulation, mRNA expression, protein localization, and cell viability. The potential effect of readthrough was also analyzed by in silico predictions. Results showed that fibroblasts from 5/11 patients exhibited an up to 3-fold increase of enzymatic activity after gentamicin treatment. Accordingly, cell lines tested showed enhanced well-localized protein and/or increased mRNA expression levels and/or reduced metabolite accumulation. Interestingly, these cell lines also showed increased enzymatic activity after PTC124 treatment, which is a PTC readthroughpromoting compound. In conclusion, our results provide a proof-of-concept that PTCs can be effectively suppressed by readthrough drugs, with different efficiencies depending on the genetic context. The screening of new compounds with readthrough activity is a strategy that can be used to develop efficient therapies for diseases caused by PTC mutations.

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Section: Glycosaminoglycans Determinationmentioning

confidence: 99%

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

et al. 2015

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“…Reaction parameters were 2 min at 95 °C followed by 45 cycles of 10 s at 95 °C, 15 s at 62 °C and 20 s at 72 °C. Results were analyzed using the 2 -ΔΔC t method [28,29] and a standard mathematical model [30].…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

DNase II deficiency impairs innate immune function in Drosophila

Seong

Varela-Ramı́rez

Aguilera

2006

Cellular Immunology

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DNase II enzymes are highly conserved proteins that are required for the degradation of DNA within phagolysosomes. Engulfment of apoptotic cells and/or bacteria by phagocytic cells requires the function of DNase II to completely destroy ingested DNA. Mutation of the dnase II gene results in an increase of undegraded apoptotic DNA within phagocytic cells in mice and nematodes. Additionally, reduction of DNase II enzymatic activity in Drosophila melanogaster has been shown to lead to increased accumulation of DNA in the ovaries. Due to the importance of DNA clearance during infection, we hypothesized that a severe reduction of DNase II activity would result in diminished immune function and viability. To test this hypothesis, we knocked down DNase II activity in flies using RNAi. As expected, expression of a dnase II-RNAi construct in flies resulted in a dramatic reduction of DNase II activity and a significant decrease in total hemocyte numbers. Furthermore, infection of dnase II-RNAi flies with Gram negative or positive bacteria resulted in a severe reduction in fly viability. These results confirm that DNase II and the ability to clear macromolecular DNA is essential for maintaining proper immune function in Drosophila.

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“…Quantitative PCR was performed with an ABI PRISM J 7700 sequence detection system (Applied Biosystems) by SYBR green assay, employing primers mSPP1-TAF 5 0 -ACTTTCACTCCAATCGTCCCTA-3 0 specific for bases 175-195 in exon 5 and mSPP1-TAR 5 0 -TGTGGCATCAGGATACTGTT-CAT-3 0 specific for bases 278 in exon 5 to bases 1-15 in exon 6. The quantity was normalized to 18s-ribosomal RNA using the formula of the 2 -DDCt method [43]. Evaluation was performed in triplicate.…”

Section: Evaluation Of Opn Mrna In Splenocytesmentioning

confidence: 99%

Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr mice

Miyazaki

Ono

et al. 2005

Eur J Immunol

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Potentially, autoimmune diseases develop from a combination of multiple genes with allelic polymorphisms. An MRL/Mp-Fas lpr/lpr (MRL/lpr) strain of mice develops autoimmune diseases, including lupus nephritis, but another lpr strain, C3H/HeJFas lpr/lpr (C3H/lpr) does not. This indicates that MRL polymorphic genes are involved in the development of the diseases. By quantitative trait loci (QTL) analysis using 527 of the (MRL/lpr Â C3H/lpr)F 2 mice, we identified a novel locus for susceptibility to lupus nephritis at map position D5Mit115 on chromosome 5, the same alias of the osteopontin (Opn) gene (LOD score =4.0), susceptible in the MRL allele. In functional analyses of the MRL and C3H Opn alleles using synthetic osteopontin (OPN) made with a new method "cell-free system" with wheat germ ribosomes, the MRL-OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF-a, IL-1b and IFN-c in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis.

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Quantitative Reverse Transcription–Polymerase Chain Reaction to Study mRNA Decay: Comparison of Endpoint and Real-Time Methods

Cited by 922 publications

References 18 publications

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

DNase II deficiency impairs innate immune function in Drosophila

Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr mice

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