Ecodry premix oligo dt

After the hAT-MSCs were co-cultured with BTICs [medulloblastoma (N = 3), AT/RT (N = 2) and glioblastoma (N = 3)], total RNA was extracted from hAT-MSCs using the PureLink RNA mini kit (Invitrogen), and cDNA synthesis was performed using EcoDry Premix-oligo dT (Clontech, Mountain View, CA) following the manufacturer’s instructions. The mRNA levels of the selected genes were assessed by real-time quantitative PCR (RT-qPCR) using custom TaqMan array plates (Applied Biosystems, Carlsbad, CA) and an ABI7500 system. Probes were selected from inventoried gene expression assays (Invitrogen, S2 Table) and customized in triplicate. RT-qPCR analysis was performed using TaqMan probes for cyto-chemokine receptors and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reactions were performed under conditions specified in the ABI TaqMan Gene Quantitation assay protocol and were repeated in triplicate. The comparative threshold cycle (Ct) method was used to calculate the relative gene expression. To quantify gene expression, the target genes were normalized to an endogenous reference (GAPDH). Relative quantification of target gene expression was calculated using the comparative Ct method. All experiments were conducted in triplicate.

Hippocampus was snap frozen in liquid nitrogen and stored at −80 °C, and total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription of total RNA (1 μg) was carried out using RNA to cDNA EcoDry Premix (Oligo dT) (Clontech) according to the manufacturer’s protocol. qPCR was performed using a Bio-Rad CFX-96 quantitative thermocycler and SsoFast EvaGreen Supermix (Bio-Rad). Relative changes in gene expression were determined using the 2^−ΔΔCt method. Primer sequences used for qPCR can be found in Supplementary Table 1.

Total cellular RNA was prepared from the cells by using and RNeasy Mini Kit (Qiagen). And total RNA was used to synthesize the first strand cDNA using RNA to cDNA EcoDry premix Oligo-dT (Clontech). Quantitative PCRs were performed with the use of SYBR green PCR Master Mix (Toyobo) [59 (link)] and we used an ABI 7300 real time PCR system (Applied Biosystems). GAPDH was used as the internal standard for normalization. The primer sequences used are as follows; SCRN1 primer set (forward: 5′-GGAGAGGGCGAGTTCAATTT-3′; reverse: 5′-GCACTGTGATGCTTTCTTCTTG-3′), GAPDH primer set (forward: 5′-GGTGTGAACCATGAGAAGTATGA-3′; reverse: 5′-GAGTCCTTCCACGATACCAAAG-3′).

Total DNAs or RNAs were extracted from cultured cells in liquid medium and transgenic calli using a DNeasy Plant Mini Kit or RNeasy Plant Mini Kit (Qiagen). Cultured cells and calli were harvested by centrifugation at 10,000 r.p.m. for 1 min. The cDNA from total RNA was reverse-transcribed using RNA to cDNA EcoDry Premix (Oligo dT) (Takara).