Page 160 - Molecular features of low-grade developmental brain tumours

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CHAPTER 6
microRNA Array. After washing, the slides were scanned and analysed using ImaGene® 9 (miRCURY LNATM microRNA Array Analysis Software, Exiqon, Vedbaek, Denmark). The quantified signals were corrected for background (Normexp with offset value 10 31 and normalized using the global Lowess (Locally Weighted Scatterplot Smoothing) regression algorithm.
MicroRNA target prediction
Using the online database miRWalk (version 2.0; http://zmf.umm.uni-heidelberg.de/apps/ zmf/mirwalk/) a total of 5 different databases (miRWalk, Microt4, miRanda, miRDB and Targetscan) were used to identify predicted targets of miR-519d and miR-4758. Targets were considered relevant if found in at least 3 of the 5 databases.
Quantitative real-time PCR analysis
miRNA (miR-519d-3p, miR-4758-3p, miR-664b-3p, miR-4714-5p, miR-5681b and a reference gene, U6B small nuclear RNA gene, Rnu6B) expression was analyzed using Taqman microRNA assays (Applied Biosystems, Foster City, CA, USA). cDNA was generated using the Taqman MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, and the PCRs were run on a Roche Lightcycler 480 thermocycler (Roche Applied Science, Basel, Switzerland). To evaluate miRNA targets (TIMP2, AKT3, ERBB3, ERBB4, PI3KCA, TP53, MDM2, CDK4, RB1, CDK2, CDKN1A (P21), CDKN1B (P27), JAK1 and PTEN), 1 μg of cell culture derived total RNA or 500 ng of human brain material-derived total RNA were reverse-transcribed into cDNA using oligo dT primers (supplementary table 1). EF1α was used as a reference gene. PCRs were run as described previously 32 on a Roche Lightcycler 480 thermocycler (Roche Applied Science, Basel, Switzerland).
Quantification was performed using the computer program LinRegPCR in which linear regression on the Log (fluorescence) per cycle number data is applied to determine the amplification efficiency per sample 33,34. The starting concentration of each specific product was divided by the starting concentration of reference genes and this ratio was compared between groups.
Cell transfection
The human pediatric low grade astrocytoma cell line (WHO grade II: Res-259; LGG2) was kindly provided by Dr Chris Jones (Institute of Cancer Research, Sutton, UK). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/HAM F10 (1:1) (Gibco, Life Technology) supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin and 10% fetal calf serum (FCS) in a humidifier incubator at 37°C with 5% CO2. Oligonucleotides were delivered to the cells using Lipofectamine® 2000 transfection reagent (Life Technologies, Grand Island, NY, USA) in a final concentration of 50 nM for a total of 24 hours. Cells were transfected with mimic pre-miRNA (Applied Biosystems, Carlsbad, CA, USA) for miR-519d-3p and/or miR- 4758-3p. Cells treated with lipofectamine without mimic were used as a control. Cells were washed twice with PBS before harvesting.
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