7300 real time pcr machine

Amplified cDNAs (1 ng/sample) from laser-captured macrophages were used for measurement of gene expression by qPCR (Applied Biosystems 7300 real time PCR machine), using a kit from Biorad. The sequences for the primers are given in S2 Table. All data were normalized to cyclophilin A and expressed as fold change over the control group results. For the laser-captured cell data, the results are from 3 independent samples, each representing amplified cDNA originating from the macrophages from one animal.

RNA was extracted from cells using the Aurum total RNA Mini Kit (Bio-Rad) according to manufacturer’s protocol and cDNA was synthesized using iScript (Bio-Rad). Each sample of cDNA was quantitated and diluted to an equal concentration of 10 ng/mL. The Applied Biosystems 7300 real-time PCR machine was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad). All primers, as described in Supplementary Table 4, were purchased from Invitrogen. HPRT1 was used for normalization of the genes of interest. The results were analyzed using 2^-ΔΔCT method and expressed as fold change to respective vehicle-treated controls.

RNA was extracted from patient-derived GBM cells using the Aurum total RNA Mini Kit (Bio-Rad) according to manufacturer’s protocol and cDNA was synthesized using iScript (Bio-Rad). Each sample of cDNA was quantitated and diluted to an equal concentration of 10 ng/mL. The Applied Biosystems 7300 real-time PCR machine was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad). All primers, as described in Additional file 1: Table 2, were purchased from Invitrogen. ACTB was used for normalization of the genes of interest. The results were analyzed using 2^−ΔΔCT method and expressed as fold change to respective non-treated controls.