Rt2 pcr arrays

To evaluate changes in gene expression the Qiagen RT² PCR arrays (human tumor metastasis and PI3K pathway) were used (Qiagen Inc., Valencia; CA, USA). RNA (2 μg per array) was reverse transcribed using the RT² First Strand Kit including DNA elimination procedure (Qiagen Inc., Valencia; CA, USA). Results were analyzed using the MS Excel based tool provided by Qiagen (PCR Array Analysis V4, available for download at https://www.qiagen.com/us/resources/resourcedetail?id=d8d1813e-e5ba-4d29-8fdf-07a3f4227e0a&lang=en). We used was a standard two-step SYBR green amplification cycle (95°C for 15 seconds and 60°C for 1 minute). n = 3 tumors per group, randomly chosen.

The RNA extracted from mice hippocampus was used to run GABA/glutamate (GG) and dopamine/serotonin (DS) RT² PCR arrays (Qiagen). Each sample was reverse transcribed using the RT² First-Strand kit (Qiagen), mixed with RT² qPCR Master Mix containing SYBR Green (Qiagen), and aliquoted (10 μL) into each well of the RT² Profiler™ PCR Arrays (Qiagen). Each sample was used to assess the expression of 168 genes related to neurotransmission. Samples were analyzed in the mouse GG (PAMM-152) and DS (PAMM-158) neurotransmitter systems by RT² Profiler™ PCR arrays following the supplier’s instructions (SABioscience/Qiagen). The arrays were run on a QuantStudio 6 flex Real-Time PCR System (Applied Biosystems by Thermo Fisher). The RT² Profiler™ PCR Array Data Analysis software package (version 3.5, Qiagen) used 2^−(ΔΔCT)-based fold change calculations [38 (link)] and a modified Student’s t test to compute two-tail, equal variance P values. Data were normalized to the housekeeping genes actin beta (Actβ), glucuronidase beta (Gusβ), heat shock protein 90 alpha (cytosolic) class B member 1 (Hsp90ab1), and Gapdh.