Page 133 - Molecular features of low-grade developmental brain tumours

DYSREGULATION OF MMP/TIMP IN SEGA: MODULATION BY MIR-320D IN VITRO
Biosystems, Foster City, CA, USA). cDNA was generated from 50 ng of RNA using TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). RT-qPCR was performed on a Lightcycler 480 Real-Time PCR System (Roche Applied Science, Indianapolis, IN, USA). Quantification of the data was done using the LinRegPCR program in which linear regression on the Log(fluorescence) per cycle number data is applied to determine the amplification efficiency per sample 61. The expression of each specific product was divided by the geometric mean of the concentration of two reference genes (mRNA: Elongation factor 1-alpha (EF1α) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH); miRNA: the U6B small nuclear RNA gene (RNU6B) and RNU44).
Immunohistochemistry
Sections were deparaffinated in xylene (3x) and rinsed in ethanol (100%, 95% and 70%). Antigen retrieval was performed by incubating the sections in 0.1M sodium citrate buffer pH 6.0 at 121°C for 10 min using a pressure cooker. Sections were washed with phosphate buffered saline (PBS, pH 7.4) after which they were blocked with 10% normal goat serum (NGS) for 30 minutes at room temperature. After removal of NGS, primary antibody (1:200 rabbit polyclonal IgG anti-MMP2, Abcam, Cambridge, UK; 1:500 mouse monoclonal IgG 3 /κ anti-MMP14, Merck, Darmstadt, Germany) in Normal Antibody Diluent (Immunological, Duiven, The Netherlands) was added to the sections and incubated overnight at 4 °C.
Figure 2. Gene expression MMPs and TIMPs differentially expression in SEGA compared to controls. a-l. Gene expression was studied using RT-qPCR analysis of MMP2 (a), MMP11 (b), MMP14 (c), MMP15 (d), MMP19 (e), TIMP1 (f), TIMP2 (g), TIMP3 (h), MMP17 (i), MMP16 (j), MMP25 (k), TIMP4 (l) in SEGA (n=12) and controls (n=8). Data are expressed relative to the expression observed in control tissue.
*p-value<0.05, **p-value<0.01, ***p-value<0.001, Mann-Whitney U test.
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