High capacity rt pcr kit

Total RNA was extracted from the liver after perfusion with PBS and preserved in RNA (Ambion, Austin, TX, USA) until the extraction of RNA. Extraction of RNA was performed by the TRIzol reagent (Life Technologies, Carlsbad, CA) or RNeasy Micro Kit (Qiagen, Hilden, Germany), according to the manufacturer's procedure. Synthetization of the first strand of complementary DNA was performed by the High capacity RT-PCR Kit (Applied Biosystems, Carlsbad, CA). Quantitative polymerase chain reaction (qPCR) was performed to measure levels of mRNA using TaqMan gene expression kit (Applied Biosystems). A VIC®-labeled probe for Gapdh was used for normalization.

Single cell suspensions were made from spleens of WT, X-MAID, or MKO male mice. 1 WT, 1 MKO, and 4-5 X-MAID spleens were used for each experiment. Cells were enriched by negative selection using rat anti-CD8 hybridoma supernatant (Clone 53-6.7) and anti-MHC II (BioXCell) antibodies followed by anti-rat magnetic beads (Qiagen). CD4⁺ T cells were further purified by flow sorting using CD4-APC (Biolegend) with the FACSJazz (BD). RNA isolation was completed using the PureLink RNA mini kit (Invitrogen). Equal amounts of RNA were reverse transcribed from each group using the High Capacity RT-PCR kit (Applied Biosystems). The resulting cDNA was used for qPCR using Msn, Ezr, and Gapdh PrimeTime qPCR primers (IDT) and SYBR Green mastermix (Applied Biosystems). qPCR Ct was quantified on the 7500 Standard (Applied Biosystems). Data are represented as ΔCT^-1 defined as the inverse of Ct of the primer of interest minus the Gapdh Ct for that sample. Technical duplicates were run for each sample.