Rapid Quantitative PCR Determination of Relative Gene Expression in Tumor Specimens Using High-Pressure Liquid Chromatography

Odin, Elisabeth; Larsson, Lars; Aram, Maddi; Gustavsson, Bengt; Larsson, Per‐Göran

doi:10.1159/000030004

Cited by 7 publications

(3 citation statements)

References 19 publications

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“…We chose the alginate system as a well-defined three-dimensional chondrocyte culture model [2,3]. The housekeeping gene ß-actin was used as an internal standard, since it is assumed to be constantly regulated in tissue [16].…”

Section: Discussionmentioning

confidence: 99%

Interleukin-1β Induces Different Gene Expression of Stromelysin, Aggrecan and Tumor-Necrosis-Factor-Stimulated Gene 6 in Human Osteoarthritic Chondrocytes in vitro

Stöve

Huch²,

Günther³

et al. 2000

Pathobiology

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Objective: To analyze the gene expression of osteoarthritic chondrocytes cultured in alginate after stimulation with interleukin (IL)-1β. Methods: Chondrocytes were isolated from osteoarthritic cartilage obtained during total knee replacement by sequential enzymatic digestion. After suspension in alginate, cells were cultured with and without 100 pg/ml IL-1β. Quantitative RT-PCR reaction was used to estimate the messenger RNA (mRNA) of three different metabolites [tumor-necrosis-factor-stimulated gene 6 (TSG-6), stromelysin-1 (MMP-3) and aggrecan (AGG)]. Results: After having shown the precision of quantitative PCR, this method allowed us to detect IL-1β-induced changes in mRNA of TSG-6, MMP-3 and AGG. MMP-3 was found to be the most abundant transcript, IL-1β induced a 12-fold upregulation of MMP-3 levels compared to control, and 7-fold of TSG-6. The AGG transcript level, indicating anabolic events, was found to be downregulated by between 2- and 3-fold. Conclusions: In our culture system, the response of osteoarthritic chondrocytes to IL-1β is preserved. Therefore, this system might be helpful for further investigation of the influences of drugs, cytokines and growth factors, for example, on the metabolism of chondrocytes at the level of gene transcription as the most basic level of regulation.

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Section: Discussionmentioning

confidence: 99%

Interleukin-1β Induces Different Gene Expression of Stromelysin, Aggrecan and Tumor-Necrosis-Factor-Stimulated Gene 6 in Human Osteoarthritic Chondrocytes in vitro

Stöve

Huch²,

Günther³

et al. 2000

Pathobiology

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“…␤-Actin is a well-known housekeeping gene and has been commonly used as an internal control for the RT-PCR studies. [25][26][27][28] As mRNA of ␤-actin exists in both the viable epithelial and dermal cells, in this series we just simply excised the epithelial part of the keloid for all the specimens used in RT-PCR studies. In this circumstance, identification of the ␤-actin fragments in the RT-PCR study mainly indicated the existence of viable dermal cells.…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of ␤-actin cDNA was an internal control for the immersed skin organ culture model and PCR reactions. [25][26][27][28] Primers for ␤-actin cDNA were ␤A-1 (5'-CTCTTCCAGCCTTCCTTCC-3') and ␤A-2 (5'-GTCACCT-TCACCGTTCCAG-3'), which generated a 559-bp PCR product. The cycling parameters were 5 minutes at 94°C, followed by 35 cycles of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C.…”

Section: Rt-pcr Amplification Of Aav Lacz and ␤-Actin Mrnamentioning

confidence: 99%

Gene Transfer into Human Keloid Tissue with Adeno-Associated Virus Vector

Cheng

et al. 2003

The Journal of Trauma: Injury, Infection, and Critical Care

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BACKGROUND Gene transfer is a new territory for clinicians. Intractable disorders might be approached in such a way. Adeno-associated virus (AAV) vector has been transfected successfully into a variety of tissues including skin. We evaluated the ability of this vector to transfer and cause expression of the reporter gene in human keloid tissue. METHODS Human keloid specimens were injected with an AAV vector encoding beta-galactosidase and incubated for 4 weeks after injection. The presence of mRNA and beta-galactosidase enzymatic activity were assayed by reverse-transcriptase polymerase chain reaction and the X-gal technique. RESULTS Gene expression shown by reverse-transcriptase polymerase chain reaction was observed in keloid tissue 4 weeks after injection, and so was the positive X-gal staining. CONCLUSION Our results showed that AAV vector could transduce human keloid tissue effectively. Replacement of the reporter gene with a functioning gene might be feasible for keloid treatment.

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Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection

Odin

Wettergren

Larsson

et al. 1999

Journal of Chromatography B: Biomedical Sciences and Applicatio

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Rapid Quantitative PCR Determination of Relative Gene Expression in Tumor Specimens Using High-Pressure Liquid Chromatography

Cited by 7 publications

References 19 publications

Interleukin-1β Induces Different Gene Expression of Stromelysin, Aggrecan and Tumor-Necrosis-Factor-Stimulated Gene 6 in Human Osteoarthritic Chondrocytes in vitro

Interleukin-1β Induces Different Gene Expression of Stromelysin, Aggrecan and Tumor-Necrosis-Factor-Stimulated Gene 6 in Human Osteoarthritic Chondrocytes in vitro

Gene Transfer into Human Keloid Tissue with Adeno-Associated Virus Vector

Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection

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