Laser‐assisted microdissection applied to frozen surgical pathologic specimens – methodological aspects on RT‐PCR

Saal, Isabelle; Gustin, A.; Rombaut, Katia; Pelaez, Pilar; Rorive, Sandrine; Remmelink, Myriam; Salmon, Isabelle

doi:10.1111/j.1533-869x.2003.01106.x

Cited by 3 publications

(4 citation statements)

References 19 publications

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“…We used a procedure that we have detailed elsewhere (18). Briefly, a human glioblastoma was obtained immediately after surgery from the Department of Neurosurgery of the Erasmus University Hospital and directly submerged in RNAlater (Westburg).…”

Section: In Vivo Stereotactic Procedures and Treatmentsmentioning

confidence: 99%

“…This procedure is specifically recommended for first-strand cDNA synthesis using less than 50 ng of RNA. The reaction was performed in a total volume of 20 L containing 2 L of supplied 10ϫ concentrated buffer, 1 L of oligo(dT) [12][13][14][15][16][17][18] …”

Section: In Vivo Stereotactic Procedures and Treatmentsmentioning

confidence: 99%

“…We therefore performed a cDNA microarray profiling of the interleukins (ILs) and IL receptors (IL-Rs) in HUVECs and GBM microvessels (obtained by means of a laser-assisted microscope system; ref. 18) to investigate whether HUVECs can be used as an in vitro model to characterize potential proangiogenic effects mediated by gastrin.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

Lefranc¹,

Mijatovic

Mathieu

et al. 2004

Clinical Cancer Research

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Purpose: This study aims to investigate the role of gastrin-17 (G17) on angiogenesis features in gliomas both in vitro and in vivo.Experimental Design: The influences of G17 and G17 receptor antagonists were characterized in vitro in terms of angiogenesis on human umbilical vein endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with respect to U373 orthotopic glioma xenografts. The influence of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor-B inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis. G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced modifications in nuclear factor-B DNA binding activity were characterized by means of specific enzyme-linked immunosorbent assays. The influence of G17 on E-and Pselectin expression was determined by means of computerassisted microscopy, whereas the influence of E-and P-selectin on HUVEC migration was approached by means of antisense oligonucleotides. The chemotactic influence of G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted videomicroscopy with Dunn chambers.Results: Messenger RNAs for cholecystokinin (CCK) A , CCK B , and CCK C receptors were present in HUVECs and microvessels dissected from a human glioblastoma. Whereas G17 significantly increased the levels of angiogenesis in vivo in the U373 experimental glioma model and in vitro in the HUVECs, the CCK B receptor antagonist L365,260 significantly counteracted the G17-mediated proangiogenic effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor ␣ (CXCR1) and IL-8 receptor ␤ (CXCR2) mRNAs were not detected in these endothelial cells. Gastrin significantly (but only transiently) decreased the level of expression of E-selectin, but not P-selectin, whereas IL-8 increased the expression of E-selectin. Specific antisense oligonucleotides against E-and P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on Matrigel.Conclusions: The present study shows that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on HUVECs. This effect depends in part on the level of E-selectin activation, but not on IL-8 expression/ release by HUVECs.

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Section: In Vivo Stereotactic Procedures and Treatmentsmentioning

confidence: 99%

Section: In Vivo Stereotactic Procedures and Treatmentsmentioning

confidence: 99%

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Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

Lefranc¹,

Mijatovic

Mathieu

et al. 2004

Clinical Cancer Research

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“…Este último reagente permite a extração inicial do RNA, seguida pela extração de DNA Mais recentemente, tem sido extraído RNA de apenas uma célula, empregando-se a técnica de microdissecção a laser. 47 O método da RT-PCR consiste na combinação da técnica de transcriptase reversa com a PCR, envolvendo três etapas:…”

Section: Rt-pcr (Reverse Transcription-pcr)unclassified

Biologia molecular aplicada às dermatoses tropicais

Roselino

2008

An. Bras. Dermatol.

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São apresentados conceitos básicos sobre célula, código genético e síntese protéica, e sobre algumas técnicas de biologia molecular, tais como PCR, PCR-RFLP, seqüenciamento de DNA, RT-PCR e immunoblotting. São fornecidos protocolos de extração de nucleotídeos e de proteínas, como salting out no sangue periférico e métodos do fenol-clorofórmio e do trizol em tecidos. Seguem-se exemplos comentados da aplicação de técnicas de biologia molecular para o diagnóstico etiológico e pesquisa em dermatoses tropicais, com ênfase na leishmaniose tegumentar americana e hanseníase.

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Role of Myeloperoxidase in ROS Generation and Inflammation Response on Prostate Epithelial Cells

et al. 2023

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Myeloperoxidase (MPO) has been reported in prostate tissue, and considering its pro-oxidant properties, this location might be linked to prostate pathology. The possibility that the glandular prostatic tissue might be the source of MPO and its potential inflammatory effects must be tested. Human prostate material was obtained from prostate biopsies and radical prostatectomies. Immunohistochemistry was performed using MPO-specific human antibody. In situ hybridization using MPO-specific probes and laser-assisted microdissection for quantitative real-time RT-PCR were performed to observe whether MPO is being produced in prostate tissue. Mass spectrometry on prostate biopsies was used to detect products of MPO activity in nucleic acids (DNA/RNA). MPO contribution to intracellular accumulation of ROS and interleukin-8 in prostatic epithelial cells was monitored in vitro. Immunohistochemistry confirmed cellular localization of MPO in epithelial cells of the prostate. The staining varied from light to high intensity. In situ hybridization did not address the presence of mRNA coding for MPO. No MPO-specific modifications on nucleic acids were detected. Mox-LDL was a major factor inducing ROS and cytokines production in prostatic epithelial cells. We did not demonstrate that MPO was synthetized by prostatic epithelial cells. However, in vitro experiments showed the ability of MPO to potentiate the ROS production and inflammation on prostate epithelial cells. Results do not allow us to demonstrate a role of MPO in prostate to date but further studies are mandatory to focus on the potential impact of MPO in the development of prostatic diseases.

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Laser‐assisted microdissection applied to frozen surgical pathologic specimens – methodological aspects on RT‐PCR

Cited by 3 publications

References 19 publications

Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

Biologia molecular aplicada às dermatoses tropicais

Role of Myeloperoxidase in ROS Generation and Inflammation Response on Prostate Epithelial Cells

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