Topreal qpcr 2x premix taqman probe

RNA from Th cells cultured for 3 days was isolated using Trizol reagent (Molecular Research Center, Cincinnati, OH, USA). Next, cDNA was synthesized using TopScript RT (Enzynomics), and a qRT-PCR was performed using TOPreal™ qPCR 2X PreMIX SYBR green (Enzynomics Daejeon, Republic of Korea), TOPreal™ qPCR 2X PreMIX Taqman probe (Enzynomics Daejeon, Republic of Korea), and a Roche LightCycler 96. The primers used for qRT-PCR are listed in the Supplementary Information Table S1.

The hippocampi (n = 8 mice/group) were collected and digested in Trizol reagent for RNA extraction based on the manufacturer’s instructions (RNAeasy Lipid Tissue Mini Kit; Qiagen). Reverse transcription was performed using the Superscript™ II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA). The resulting complementary DNA (cDNA) was diluted with RNase-free water to achieve a final concentration of 8 ng/μL, and the samples were stored at −70 °C. The quantitative PCR was performed with TOPreal™ qPCR 2XPreMIX (TaqMan Probe) (Enzynomics, Daejon, Korea) using the LineGene 9600 Plus machine (BIOER, Hangzhou, China) according to the manufacturer’s instructions. The qRT-PCR primers are listed in Supplementary Table S3. The annealing temperature for the reaction was 58.5 °C, and the built-in software generated the amplification curves and computed the threshold cycle values. All the readouts were normalized using the β-actin reference gene. Via the 2^−ΔΔCT method [64 (link)], data were expressed as mean relative values compared to the values of the WT controls.