Mastercycler realplex4 real time pcr detection system

Total RNA was extracted with the RNeasy Micro Kit (Qiagen, 74004) and RNeasy Mini Kit (Qiagen, 74106). Complementary DNA (cDNA) was synthesized from the extracted RNA with the iScript cDNA Synthesis Kit (Bio-rad, 170-8891). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays were performed with Thermo Scientific Solaris qPCR Rox Master Mix (AB-4351/C) or with Power SYBR green PCR Master Mix (Applied biosystems, Cat. No. 43091655) with a Mastercycler realplex4 real-time PCR detection system (Eppendorf), according to the manufacturers’ protocols. The abundances of the mRNAs of interest in each sample were normalized to that of ACTB/Actb or HPRT1/Hprt mRNA, and fold-changes in the abundances of target mRNAs relative to their basal abundances were calculated with the 2^−ΔΔC_t method. The primers and probes for human and mouse IRAK1/Irak1, MAP3K7/Map3k7, TAB2/Tab2, NFKBIE/Nfkbie, MAP3K8/Map3k8, MAPK3/Mapk3, MAPK14/Mapk14, IRF9/Irf9, IRAK2/Irak2, IRAK4/Irak4, CD14/Cd14, IKBKB/Ikbkb, MAPK1/Mapk1, CREBBP/Crebbp, EP300/Ep300, TNF/Tnf, NFKBIZ/Nfkbiz, and IL6/Il6 were purchased from GE Healthcare, Dharmacon (Solaris Mouse or Human qPCR Gene Expression Assay).

Total RNA was isolated from approximately 6.4 × 10⁵ cells with an RNAeasy Mini Kit (Qiagen). Quantitative RT-PCR analyses were performed with the iScript cDNA kit (Bio-RAD) and Solaris qPCR gene-specific probe sets and master mix (Thermo Fisher) with a Mastercycler realplex4 real-time PCR detection system (Eppendorf), according to the manufacturers’ protocols. The abundances of the mRNAs of interest in each sample were normalized to that of β-actin mRNA, and fold-changes in target mRNAs relative to their basal abundances were calculated by the 2^−ΔΔCt method.