Measurement of Sputum <i>Mycobacterium tuberculosis</i> Messenger RNA as a Surrogate for Response to Chemotherapy

DesJardin, Lucy E.; Perkins, Mark D.; Wolski, Kathy; Haun, Shirley; Teixeira, Lucileia; Chen, Ying; Johnson, John L.; Ellner, Jerrold J.; Dietze, Reynaldo; Bates, Joseph H.; Cave, M. Donald; Eisenach, Kathleen D.

doi:10.1164/ajrccm.160.1.9811006

Cited by 113 publications

(89 citation statements)

References 32 publications

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“…More recently, the emergence of molecular medicine has resulted in the increased use of techniques able to quantitate levels of RNA in clinical diagnostics. Such applications are wide-ranging and include procedures designed to quantify the regulation and expression of drug resistance markers in tumour cells (Ramachandran & Melnick 1999), monitor responses to chemotherapy (Desjardin et al 1999), measure the biodistribution and of transcription of geneencoded therapeutics (Fairman et al 1999), provide a molecular assessment of tumour stage (Bustin & Dorudi 1998), detect circulating tumour cells in cancer patients (Ghossein & Rosai 1996), and detect bacterial (Hill 1996) and viral (Holodniy 1994) pathogens.…”

Section: Why Mrna Quantification?mentioning

confidence: 99%

“…Similarly, the calcium ionophore A23187 induces GAPDH transcription through specific motifs in its promoter (Chao et al 1990). Growth hormone (Freyschuss et al 1994), vitamin D (Desprez et al 1992), oxidative stress (Ito et al 1996), hypoxia (Graven et al 1994, Zhong & Simons 1999, manganese (Hazell et al 1999) and the tumour suppressor TP53 (Chen et al 1999), have all been shown to activate its transcription, which is also inducible in endothelial cells (Rimarachin et al 1992) and during apoptosis (Ishitani et al 1997). Furthermore, it has been shown to be upregulated in cancer -for example in rat hepatomas (Chang et al 1998), malignant murine cell lines (Bhatia et al 1994) and human prostate carcinoma (Ripple & Wilding 1995).…”

Section: Gapdhmentioning

confidence: 99%

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Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,424

2,448

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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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Section: Why Mrna Quantification?mentioning

confidence: 99%

Section: Gapdhmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,424

2,448

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“…These sequences were as follows: RT primer (-266RT) 5¢-gcgtgtgctcgtcg-3¢; Forward primer (160F) 5¢-gcggtggtccgtttgga-3¢; Reverse primer (227R) 5¢-ccagggtcaagctcgacgtt-3¢; Probe (181T) 5¢-FAMcccggcattgacgtcgacaagg-3¢TAMRA. Sequences used to detect M. tuberculosis acr1 and 16S were as described previously (Desjardin et al, 1999;2001). All probes were dually labelled with 5 carbofluorescein (FAM) at the 5¢ end and N,N,N¢,N¢-tetramethyl-6-carborhodamine (TAMRA) at the 3¢ end.…”

Section: Resistance To Hydrogen Peroxidementioning

confidence: 99%

The stress‐responsive chaperone α‐crystallin 2 is required for pathogenesis of Mycobacterium tuberculosis

Stewart

Newton

Wilkinson

et al. 2005

Molecular Microbiology

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SummaryMycobacterium tuberculosis has two members of the a a a a -crystallin (Acr) family of molecular chaperones. Expression of Acr1 is induced by exposure to hypoxia or nitric oxide and is associated with bacterial persistence in a non-replicating state. Expression of Acr2 is induced by heat shock, oxidative stress, and uptake by macrophages. We have shown that Acr2 continues to be expressed at a high level during both acute and chronic infection in the mouse model, with an increased ratio of acr2 : acr1 mRNA in the persistent phase. Deletion of the acr2 gene resulted in a decrease in the resistance of M. tuberculosis to oxidative stress but did not impair growth in mouse bone marrow macrophages. There was no difference in bacterial load in mice infected with an acr2 deletion mutant, but a marked alteration in disease progression was evident from reduced weight loss over a prolonged infection. This correlated with reduced recruitment of T-cells and macrophages to the lungs of mice infected with the acr2 mutant and reduced immune-related pathology. These findings demonstrate that both a a a a -crystallins contribute to persistent infection with M. tuberculosis and suggest that manipulation of acr expression can influence the host response to infection.

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“…Applications of real-time RT-PCR include quantifying the regulation and expression of drug resistance markers in tumor cells (Ramachandran and Melnick, 1999), monitoring responses to chemotherapy (Desjardin, Perkins, Wolski, et. al, 1999), providing a molecular assessment of tumor stage (Bustin and Dorudi, 1998), detecting circulating tumor cells in cancer patients (Ghossein and Rosai, 1996), and detecting bacterial (Hill, 1996) and viral (Holodniy, 1994) pathogens including HIV (Connor et al…”

Section: 3b Gene Expression (Mrna Quantification)mentioning

confidence: 99%

Real-Time PCR Measurement by Fluorescence Anisotropy

et al. 2005

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Real-time polymerase chain reaction (PCR) is the gold-standard for quantitation in both mutation and gene expression analyses. Already this technique has found valuable clinical application in disease diagnosis and progression evaluation. As the number of known gene-disease correlations continues to rise, there will be increased demand for higher throughput and decreased cost for these analyses. Present real-time PCR measurement is based upon the fluorescent intensity of either intercalating dyes or oligonucleotide probes. Intercalating dye methods suffer from a lack of binding specificity, while probe methods are expensive and require increased assay optimization.In this thesis, a new method is presented for monitoring real-time PCR that utilizes the fluorescent anisotropy (FA) of labeled primers. FA, when measured at constant temperature, is indicative of the molecular mass to which the fluorophore is attached. Specificity is improved with the FA method over the use of intercalating dyes since the selective binding of primers is required for signal change. Assay complexity and cost are reduced compared to fluorogenic probe methods since the probes are eliminated.The design of a prototype instrument, which successfully implements this new method, is presented. Instrument and assay performance are compared to intercalating dye assays run in commercially available instrumentation. Theoretical limits on performance are also presented and compared to experimental results. Excellent repeatability and linearity are observed with respect to these benchmarks. This new method, having both high specificity and low optimization complexity, is expected to be particularly applicable to the demanding robustness requirements of nano-scale PCR.

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Measurement of Sputum Mycobacterium tuberculosis Messenger RNA as a Surrogate for Response to Chemotherapy

Cited by 113 publications

References 32 publications

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

The stress‐responsive chaperone α‐crystallin 2 is required for pathogenesis of Mycobacterium tuberculosis

Real-Time PCR Measurement by Fluorescence Anisotropy

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