Ez 1 rna tissue mini kits

The F1 mice (n = 7) were sacrificed under deep pentobarbital anesthesia at the age of 15–17 week. The prefrontal cortex of each mouse was dissected and frozen in liquid nitrogen and then stored at -80 °C. Total RNA extraction was performed by EZ-1 RNA tissue mini kits and EZ-1 Advance XL (Qiagen). Purity and concentration of total RNA was examined and cDNA synthesis from total RNA was done as the experiment (1). The mRNA expression levels of 18S rRNA (internal control), NMDA receptor subunits (NR1, NR2A and NR2B) and AMPA receptor subunits (GluR1, GluR2, GluR3 and GluR4), 5-hydroxytryptamine (serotonin) receptor 5B (5-HT 5B) and brain-derived neurotrophic factor (BDNF) were determined by using real-time RT-PCR (Light Cycler 96, Roche, Germany). The primers for NR1, NM_008169; NR2A, NM_008170; NR2B, NM_008171; GluR1, NM_008165; GluR2, NM_013540; GluR3, NM_016886; GluR4, NM_019691; 5- HT 5B, NM_024395 and BDNF, NM_012513 were purchased from Qiagen Sample & Assay Technologies. The primers for 18S rRNA (forward: 5′-TACCACATCCAAAAGGCAG-3′, reverse: 5′-TGCCCTCCAATGGATCCTC-3′) was purchased from Hokkaido System Science. Data were analyzed as above.

The prefrontal cortex (PFC) was collected from each mouse of the control and arsenite exposed 74-week-old F1 female mice (n = 7 per each group) and was frozen in liquid nitrogen and then stored at −80 °C. Total RNA extraction was performed by EZ-1 RNA tissue mini kits and BioRobot EZ-1 (Qiagen GmbH, Hilden, Germany). Purity and concentration of total RNA was examined by ND-1000 NanoDrop RNA Assay protocol (NanoDrop Technologies, Wilmington, DE, USA). Then, first strand cDNA synthesis from total RNA was done by SuperScript RNase H-Reverse Transcriptase II (Invitrogen, Carlsbad, CA, USA) and thermal cycler (Gene Atlas E, ASTEC, Fukuoka, Japan). The mRNA expression levels of 18S rRNA (internal control), NFκB (nuclear factor kappa B), IL-1β (interleukin 1β), COX2 (cyclooxygenase 2), Bax (nuclear factor kappa-light-chain-enhancer of activated B cells), Caspase-3, 5HT1A (5-hydroxytryptamine (serotonin) receptor 1A), Drd2 (dopamine receptor D2) and BDNF (brain-derived neurotrophic factor) were determined by using Light Cycler 96, Roche, Germany. The primer sequences used for neurological and immunological markers were expressed in Table 1. Data were analysed by using comparative threshold cycle method. The relative mRNA expression levels were expressed as mRNA signals per unit of 18S rRNA expression.