Taqman rna to ct 1 step pcr master mix reagent kit

Liver total RNA was isolated and purified by RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s standard protocol and stored at -80°C. Nucleic acid quality and concentration were assayed with a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA). QrtPCR expression was determined with a ABI PRISM 7000 (Applied Biosystems®, Foster City, CA) using TaqMan® RNA-to-CT™ 1-Step PCR Master Mix Reagent kit (cat # 4309169), gene-specific TaqMan PCR probes and primers, and a thermal cycler protocol as follows: 48°C for 30 min, 95°C for 10min, 95°C for 0.15 min and 60°C for 1.0 min, repeated a total of 60 cycles. Specific probe and primer TaqMan® gene expression assays were from Life Technologies™ (Carlsbad, CA) to determine hepatic mRNA levels of microsomal triglyceride transport protein (Mttp), Abcg5, and Abcg8. Sample reactions (20μL total volume each) were performed in duplicate on 96 well optical reaction plates (Applied Biosystems®, Foster City, CA). The threshold cycle from each well was established by ABI PRISM 7000 SDS software (Applied Biosystems®, Foster City, CA) and QrtPCR data were normalized to the housekeeping gene 18S RNA (cat # 4310893E) for mRNA expression of Mttp, Abcg5, and Abcg8 and made relative to the control mouse group (female WT mice on control diet = 1) for final calculations.

Brain total RNA was isolated and purified with the RNeasy mini kit (Qiagen, Valencia, CA) using the manufacturer’s standard protocol. Concentration and quality of mRNA were determined by a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA). Samples were stored at −80°C. QrtPCR expression patterns were analyzed with an ABI PRISM 7000 sequence detection system (Applied Biosystems®, Foster City, CA) using TaqMan® RNA-to-C_T^™ 1-Step PCR Master Mix Reagent kit, gene-specific TaqMan PCR probes and primers. The thermal cycler protocol was as follows: 48°C for 30 min, 95°C for 10 min, 95°C for 0.15 min and 60°C for 1.0 min, repeated a total of 40 cycles. TaqMan® gene expression assays to determine brain mRNA transcript levels of the genes listed above. Two replicates of each sample reaction (20 μL total volume each) were performed on 96 well plates (Applied Biosystems®, Foster City, CA). The threshold cycle from each well was established with ABI Prism 7000 SDS software (Applied Biosystems®, Foster City, CA) and QrtPCR data normalized to the housekeeping gene 18S RNA for mRNA. Expression of Adrbk2, Arrb2, Cnr1, Cnr2, Dagla, Daglb, Faah, Mgll, Fabp3, Fabp5, Fabp7, Naah, Nape-pld, and Trvp-1 were relative to the control female mouse group.