M mlv reverse transcriptase omniscript

Total RNA was isolated from a tissue according to the manufacturer’s protocol (NucleoSpin RNA II; Machery-Nagel, Bethlehem, PA, USA). Concentration and purity were assessed by spectrophotometric analysis (Genequant-Pro, GE Healthcare, Milan, Italy). One μg of total RNA was reverse-transcribed to cDNA with M-MLV Reverse Transcriptase (Omniscript; QIAGEN, Milan, Italy) using random primers in a 20 μL reaction incubated at 25 °C for 5 min followed by 60 min at 42 °C and 5 min at 70 °C. Two hundred ng of cDNA was amplified as follows: 15 min at 95 °C, 1 min of denaturation at 94 °C, 30 s of annealing, and 30 s of extension at 72 °C for 38 cycles. Primer sequences are summarized in Table 1. Amplification products were highlighted with ethidium bromide on 1.5% agarose gel. The intensities of the bands were quantified by densitometric analysis. The used histamine receptor gene sequences are reported in Table 1.

RNA isolation and first strand synthesis were done as previously described (Albrecht et al., 2013 (link); Rehberg et al., 2014 (link)). In brief, RNA was isolated from mouse primary hippocampal neurons on DIV3, 7, 14 and 21 using Cells-to-cDNA II^TM-Cell Lysis Buffer (Ambion^®). cDNA was generated with M-MLV reverse transcriptase Omniscript (Qiagen) using oligo-dt primers and random decamer primers. Quantitative PCR was done on a StepOnePlus real-time PCR System using TaqMan reagents and TAM-labeled predesigned expression assays for ADAP (Mm00803629_m1), p65 (Mm00501346_m1) or c-Rel (Mm01239661_m1; all Thermo Scientific). Initial deuridination and denaturation (2 min 50°C, 10 min 95°C) were followed by 40 cycles of 15 s 95°C, 1 min 60°C and expression values were calculated in relation to those obtained with the VIC-labeled housekeeping gene assay for GAPDH (4352923E) in the same wells.