Development of a direct in situ RT-PCR method using labeled primers to detect cytokine mRNA inside cells

Gey, Alain; Hamdi, Safouane; Vielh, Philippe; Mehtali, Majid; Fridman, Wolf‐Herman; Tartour, Éric

doi:10.1016/s0022-1759(99)00078-2

Cited by 6 publications

(8 citation statements)

References 31 publications

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“…In situ hybridization and rtPCR belong to the specific profile. In situ PCR combined with immunohistochemical detection is frequently used as a measurement of single-cell gene activity (Gey et al 1999). Multiplex rtPCR is also effective for observing gene expression at the single-cell level (Hahn et al 2002).…”

Section: Methods Of Genomic Expression From Single Cellsmentioning

confidence: 99%

A strategy to identify genomic expression at single-cell level or a small number of cells

2005

Electro Journal of Biotech

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Recent advances in functional genomics allow us to estimate the expression of several thousands of genes in the mammalian genome. Techniques such as microarrays, expressed tag sequencing (EST), serial analysis of gene expression (SAGE), subtractive cloning and differential display (DD), and two-dimensional electrophoresis gel have been extensively used to screen and analyze parallel gene expression. Some pathological processes, for example, tumorigenesis and solid tumour growth, in which the former is derived from a single-cell and the latter has a mixed-cell problem, present new challenges to the limit of these functional genomic techniques. To fully understand the functions of cells in tumorigenesis or in heterogeneous solid tumour masses, it is essential for scientists and physicians to develop a strategy to identify genomic expression profiles for a single-cell or for small numbers of cells. In this article, we review recently developed methods that enable functional genomic analysis at the single-cell or multicell level. In addition, the paper will review different techniques of single-cell genomic expression at the level of DNA, mRNA, protein and post-translational modifications.

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Section: Methods Of Genomic Expression From Single Cellsmentioning

confidence: 99%

A strategy to identify genomic expression at single-cell level or a small number of cells

2005

Electro Journal of Biotech

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“…Finally, counterstain (Trevigen) was added for 5 min. Coverslips were applied [15][16][17][18][19][20][21][22].…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

“…This was done in order to minimize DNA synthesis from repair-replication, which might have resulted in false priming [15]. Furthermore, all samples were exposed to prolonged DNAse treatment (at least 24 h) to eliminate amplification of genomic DNA [15][16][17][18][19][20][21][22]. A high concentration (25 U) of DNAse was used.…”

Section: Pcr For Identification Of Animalsmentioning

confidence: 99%

“…The black-stained areas against the pale-gray background (counterstain) depict positive staining for Cx43. Since we were assaying for the mRNA, only cytoplasmic staining was considered positive whereas nuclear staining would represent false-positive staining due to false priming, contamination, or DNA breakage and repair [15][16][17][18][19][20][21][22]. Figure 2a depicts in situ RT-PCR results for kidneys obtained from the WT.…”

Section: Pcr For Identification Of Animalsmentioning

confidence: 99%

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Renal morphology in connexin43 knockout mice

Silverstein

Urban

Gao

et al. 2001

Pediatric Nephrology

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Connexins (Cx) are a family of proteins that constitute the intercellular membrane channels of gap junctions. These junctions permit intercellular movement of ions and other molecules between cells, a property vital to organogenesis. Cx43 is a member of the family of channel-forming proteins that are essential for cell-cell communication of developmental signals. Studies demonstrate that Cx43 is observed in mesenchymal cells of 12-day gestation mouse kidney, a crucial period of renal development. In order to study the significance of Cx43 on renal developmental morphology, we evaluated the kidneys of embryos lacking the gene encoding for Cx43. Polymerase chain reaction (PCR) from tail specimens identified wild-type (WT), heterozygote (HT) and knockout (KO) progeny. In situ RT-PCR displayed abundant Cx43 staining in glomeruli, vasculature, and tubules in kidneys obtained from WT progeny. In contrast, Cx43 expression was completely absent in kidneys isolated from the KO. Renal histology in all three groups displayed no significant differences. Renal size was similar and there was no evidence of dysplasia or cyst formation in the KO. Our results indicate that absence of Cx43, heretofore considered essential for renal development, does not affect early renal morphological development.

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“…The IFNγ-and IL-10-producing CHO cell line was derived from the CHO-IFNγ cell line. 71 This cell line was co-transfected with a pcDNA3.1 plasmid encoding for human IL-10 and a neomycin resistance gene obtained from P. Van der Bruggen (Institut Ludwig, Brussels). The plasmid was first linearized with Sca1 A and 100 μg of this plasmid were then electroporated at 300 V and 150 μFarads (GenePulser II, Biorad).…”

Section: Discussionmentioning

confidence: 99%

Clinical validation of IFNγ/IL-10 and IFNγ/IL-2 FluoroSpot assays for the detection of Tr1 T cells and influenza vaccine monitoring in humans

Chauvat

Benhamouda

Gey

et al. 2013

Human Vaccines & Immunotherapeutics

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Development of a direct in situ RT-PCR method using labeled primers to detect cytokine mRNA inside cells

Cited by 6 publications

References 31 publications

A strategy to identify genomic expression at single-cell level or a small number of cells

A strategy to identify genomic expression at single-cell level or a small number of cells

Renal morphology in connexin43 knockout mice

Clinical validation of IFNγ/IL-10 and IFNγ/IL-2 FluoroSpot assays for the detection of Tr1 T cells and influenza vaccine monitoring in humans

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