Cloned amv first strand cdna synthesis kit

400 ng of total RNA was processed on the MyCycler Thermal Cycler (Bio Rad) for complementary DNA synthesis with the Cloned AMV First-Strand cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific) according to manufacturer instructions. All primers and cDNA samples were diluted 1/10, loaded in triplicates with SYBR Green Supermix (Bio Rad) to a 384-well PCR plate and ran on the CFX 384 Thermal Cycler (Bio Rad). Results were normalized to the reference gene nicotinamide adenine dinucleotide dehydrogenase (NADH). Primer sequences were summarized in Table 1. Primer specificity was verified by melting curve analysis. Data are presented as the percent change from controls (MF groups).

On PD3, 48 h after viral protein injections, pups were sacrificed by rapid decapitation and the brain was removed from the cranial vault for microdissection. This 48 h interval was selected on the basis of our prior study of Tat protein injection and the temporal relationship to reactive astrocytosis and protein oxidation (Aksenov et al., 2003 (link)) and early behavioral sequelae (Fitting et al., 2008b (link)). After removal of obstructing meninges the midbrain was dissected out of the surrounding midbrain tissue to facilitate access to the hippocampus. Both left and right hippocampal tissue were collected from all 6 males of each litter, and pooled within the appropriate treatment groups to ensure sufficient tissue mass for RNA isolation. Total RNA was isolated using the RNAqueous-4PCR kit (Ambion, Austin, TX) and concentration was measured by the 260/280 nm absorbance ratio (DNA Calculator, GeneQuant, Piscataway, NJ). A cDNA library was generated by the reverse transcription PCR (RT-PCR) of 1 μg purified RNA using 50 ng/μL random hexamer primers (Cloned AMV First-Strand cDNA Synthesis kit, Invitrogen, Carlsbad, CA). An additional sample lacking the reverse transcription enzyme was included in the RT-PCR reaction to control for potential DNA contamination.