Goscript cdna reverse transcription kit

Mouse hippocampal endothelial cells were isolated as described above [36 (link)]. Total RNA from the hippocampal endothelial cells was isolated using RNAiso Plus (Sangon Biotech, Shanghai, China). cDNA was synthesized using a GoScriptTM cDNA Reverse Transcription Kit (Promega, Madison, WI, USA). The expression of specific mRNAs was assayed using fluorescence-based real-time quantitative PCR (RT-PCR). Quantitative PCRs were performed using TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) in triplicate for each sample. The details of the primer sequences are as follows:
Cdk5 (forward), 59-CAATGCAGAAATACGAGAAACTGG-39;
Cdk5 (reverse), 59-CTTTGAGTAGACAGATCTCCCG-39;
Cxcl1 (forward), 59-ACCGAAGTCATAGCCACACTC-39;
Cxcl1 (reverse), 59-CTCCGTTACTTGGGGACACC-39.

Total RNA was extracted separately from the meninges and hippocampi using TRIzol reagent (Sangon Biotech, Shanghai, China). Messenger (m)RNA (1 μg) was converted into cDNA using a GoScriptTMcDNA Reverse Transcription Kit (Promega, Madison, WI, USA). The expression of specific mRNAs was assayed using fluorescence-based real-time quantitative PCR (RT-PCR). Quantitative PCR reactions were performed using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China) in triplicate for each sample. β-Actin was chosen as the reference gene because of its stable expression in the meninges and hippocampi. The amplification cycles were 95 °C for 10 s and 60 °C for 30 s; the cycle number is 40, with the melt curve at 60–95 °C. At the end of the assay, a melting curve was constructed to evaluate the specificity of the reactions. All of the quantitative real-time PCR reactions were determined and analyzed using a Bio-Rad IQ5 Real-Time PCR System with the comparative Ct method and were normalized to β-actin. The list of primers used is presented in Additional file 1: Table S1.