Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets

Benavides, Gina; Hubby, Bolyn; Grosse, W M; McGraw, Royal A.; Tarleton, Rick L.

doi:10.1016/0022-1759(94)00339-x

Cited by 36 publications

(17 citation statements)

References 20 publications

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“…The staining of cytokine-producing cells could be blocked with excess unlabeled antibodies but not with control antibodies (data not shown). Preliminary experiments also suggested that (1) the earliest immunocytochemical detection of cytokine-producing cells occurred at approximately the same point in infection that cytokine mRNA was detectable by RT-PCR [22], and (2) immunocytochemistry was more sensitive than ELI-SPOT techniques (data not shown).…”

Section: Resultsmentioning

confidence: 86%

Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Zhang

Tarleton

1996

Eur J Immunol

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Cytokine production in the spleens of mice infected with the protozoan parasite Trypanosoma cruzi was analyzed in three models which differ in the outcome of the infection. Using immunocytochemical techniques to detect cytokine-producing cells, the production of type 1 [interleukin-2 (IL-2) and interferon (IFN)-gamma], type 2 (IL-4, IL-5, IL-10), inflammatory [tumor necrosis factor (TNF)-alpha, IL-1 alpha, IL-6] and regulatory (transforming growth factor-beta) cytokines were examined. With the exception of IL-4 and IL-5, cells producing all of the cytokines assayed were detected in both the resistant and susceptible models of T. cruzi infection. Cells producing IL-4 and IL-5 were not detected until later in infection in the resistant mice (> 34 days), at about the time animals of the susceptible strain succumb to the infection. Mice of the susceptible model showed a slight delay in the appearance of cells producing the type 1 cytokines IL-2 and IFN-gamma and an earlier appearance of TNF-producing cells, in comparison to resistant mice. Cells producing IL-2 or IL-10 were transient in their appearance in the spleen while cells producing IL-1, IL-4, IL-5, IL-6, IFN-gamma, TNF, or TGF-beta were first detectable in either the acute or post-acute stage of the infection and persisted up to 700 days post infection in two different resistant models of the infection. Cells producing IFN-gamma, TNF-alpha and TGF-beta were particularly numerous even very late in the infection. Double-staining techniques were used to show that the vast majority of the IFN-gamma-producing cells in the spleen were CD4-, CD8- alpha/beta TCR+T cells. This study confirms the transience of IL-2 production in the acute stage of T. cruzi infection and the persistent and simultaneous production of type 1 and type 2 cytokines during the late-acute and chronic stages of the infection. Susceptibility or resistance to T. cruzi infection does not associate with a Th2 pattern of cytokine production in the three models examined in this study. The overlapping pattern of type 1 and type 2 cytokine-producing cells in both the acute and chronic stages of T. cruzi infection demonstrates that longterm infections do not necessarily lead to a dominance of either type 1 or type 2 cytokine production.

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Section: Resultsmentioning

confidence: 86%

Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Zhang

Tarleton

1996

Eur J Immunol

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“…In a previous study we showed that our PCR protocol was able to detect an amount of parasite DNA equivalent to 10 fg in blood samples (Gomes et al 1998). Many other reports have described the use of T. cruzi-specific quantitative (Benavides et al 1995 ;Graefe et al 2003 ;Schijman et al 2003) 2002) PCR. Although all those methods were very sensitive and reliable to determine the amount of parasite present in biological samples, their use have been restricted to the parasite quantification in blood (Gomes et al 1998 ;Schijman et al 2003) or in tissues from acute-infected mice (Hoft and Eickhoff, 2002 ;Graefe et al 2003).…”

Section: Discussionmentioning

confidence: 94%

Comparative study of the presence of Trypanosoma cruzi kDNA, inflammation and denervation in chagasic patients with and without megaesophagus

et al. 2005

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Neuronal lesions have been considered the hallmark of chagasic megaesophagus, but the role of Trypanosoma cruzi and the participation of the inflammatory cells in this process are still debated. In the present study we counted neurons in the oesophagus from patients with and without megaesophagus and further examined these samples for the presence of parasite kDNA and cells with cytolytic potential (Natural Killer cells, cytotoxic lymphocytes and macrophages). The presence of parasite kDNA was demonstrated in 100% of cases with megaesophagus and in 60% of patients without megaesophagus. When analysed for the number of neurons, the patients without megaesophagus could be classified into 2 groups, as having normal or a decreased number of neurons. The former group did not show any inflammatory process, but interestingly, all patients without megaesophagus presenting decreased number of neurons also presented both parasite kDNA and inflammatory process in the organ. We further observed that the numbers of cytotoxic cells in the myenteric plexus region inversely correlate with the number of neurons. These data together strongly suggest that chronic lesions in chagasic megaesophagus might be a consequence of immune-mediated mechanisms, that last until the chronic phase of infection, and are dependent on the persistence of parasite in the host's tissue.

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“…This procedure provides an efficient measurement method in c-PCR (66,74,75,82). Agarose or polyacrylamide gels can be used, acrylamide in particular providing an easier substrate for the resolution of bands differing by only few base pairs.…”

Section: Methods For the Detection And Measurement Of Pcr Productsmentioning

confidence: 99%

“…An example is the approach described by Wang et al (40) for the quantification of various mRNAs encoding cytokines, by quantitative RT-PCR. This type of competitor is normally obtained from two separated sequences containing the 5' and 3' primers, respectively, separated by a spacer sequence (40,48,(65)(66)(67). This type of competitor, which can be used for DNA or RNA measurements, has the important advantage that several different measurements can be performed using the same competitor template, with a significant improvement in assay accuracy.…”

Section: Multiple Competitor Templatesmentioning

confidence: 99%

Developments in Quantitative PCR

Orlando¹,

Pinzani²,

Pazzagli³

1998

CCLM

296

154

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In recent years the growing interest in quantitative applications of the polymerase chain reaction (PCR) has favoured the development of a large number of assay procedures suitable for this purpose. In this paper we review some basic principles of quantitative PCR and in particular the role of reference materials and calibrators and the different strategies adopted for nucleic acid quantification. We focus on two methodological approaches for quantitative PCR in this review: competitive PCR and real-time quantitative PCR based on the use of fluorogenic probes. The first is one of the most common methods of quantitative PCR and we discuss the structure of the competitors and the various assay procedures. The second section is dedicated to a recent promising technology for quantitative PCR in which the use of fluorogenic probes and dedicated instrumentation allows the development of homogeneous methods. Assay performance of these methods in terms of practicability and reliability indicates that these kinds of technologies will have a widespread use in the clinical laboratory in the near future.

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Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets

Cited by 36 publications

References 20 publications

Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Comparative study of the presence of Trypanosoma cruzi kDNA, inflammation and denervation in chagasic patients with and without megaesophagus

Developments in Quantitative PCR

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