Quantstudio 7 flex real

To measure the mRNA levels of related genes, total RNA was extracted utilizing TransZol Up (TransGen Biotech, Beijing, China). Subsequently, the specimens were applied to reverse-transcribes into cDNA by the cDNA Synthesis SuperMix (Transgen, China) and EasyScript^®One-Step gDNA Removal for subsequent experiments. ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used to determine mRNA levels. The primers of HMGB1, MYD88, TLR4, NF-κB, IL-1β, IL-6, IL-18, TNF-α, NLRP3, ASC and caspase-1 were engineered by Primer Premier software. All reactions were performed employing a Quant Studio 7 Flex real-time PCR system (ABI 7900HT Applied Biosystems, Bedford, MA, USA). The primer sequences were listed in Table 1. GAPDH was regarded as a house-keeping gene, and the relative changes in gene mRNA levels were evaluated by the 2^−ΔΔCT method.

RNA was extracted using a kit (NucleoSpin® RNA Plus, MACHERY-NAGEL) according to manufacturer’s instructions. cDNA was synthesized using SuperScript™ IV Reverse Transcriptase (Invitrogen). qPCR was conducted using Power SYBR Green PCR master mix (ThermoFisher Scientific) and QuantStudio 7 flex real-time PCR system (Applied Biosystems). Supplementary Table 1b lists qPCR primers.
Total murine Prlr = LF + SF3 (SF1 and SF2 were not expressed in splenocytes and B cells). Total human PRLR = LF + IF + SF1a + SF1b. There is a huge degree of variability in the absolute expression of the isoforms across individual mice in each strain. To account for this variability and to most accurately predict changes in pro-proliferative, anti-apoptotic effects of PRLRs in cells after LFPRLR knockdown, expression of PRLR isoforms in each mouse was represented as the ratio of LFPRLR: total PRLR. An exception to calculating the ratio is made in instances where the cells express only LF/IFPRLR, as in some malignant human B cells.