Sybr green realtime pcr master kit

cDNA synthesis from mRNA was done with a ReverTra Ace qPCR RT Master Mix Kit (FSQ-201, TOYOBO, Japan) according to the manufacturers’ protocols (800ng total RNA per reaction). cDNA synthesis from miRNA was performed with a miRcute Plus miRNA First-Strand cDNA Kit (KR-211, TIANGEN) according to the manufacturer’s instructions (1μg total RNA).
Real-time qPCR was performed with a SYBR^® Green Realtime PCR Master Kit (TOYOBO) and miRcute Plus miRNA qPCR Kit (SYBR Green, TIANGEN) in a qTOWER³G device (Analytikjena, Germany). The specific PCR primers use for the detection of AchE, PSD-95 and β-actin were designed according to the NCBI sequence and synthesized by Sangon Biotech Co., Ltd. The sequences of the primers were as follows (Table 1):

Cells and tissues were fully lysed with TRIzol (15596026, Invitrogen, USA) according to manufacturer's instructions. The RNA concentration was determined by a NanoDrop instrument. A reverse transcription kit (FSQ-101, Toyobo) was used to synthesize cDNA. Real-time PCR was performed using an ABI 7500 Fast PCR instrument with the SYBR Green Real-time PCR Master Kit (QPK-201, Toyobo). The primer sequences were as follows: NLRP3 (forward: 5′-CAACCTCACGTCACACTGCT-3′; reverse: 5′-TTTCAGACAACCCCAGGTTC-3′), caspase-1 (forward: 5′-ACACGTCTTGCCCTCATTATCT-3′; reverse: 5′-ATAACCTTGGGCTTGTCTTTCA-3′), and GAPDH (forward: 5′-AAGAAGGTGGTGAAGCAGGC-3′; reverse: 5′-TCCACCACCCAGTTGCTGTA-3′). GAPDH was used to normalize target expression levels. RNA expression levels were computed by the 2^−△△CT method.

The PMs were treated with Trizol reagent (Thermo Fisher, Waltham, MA, USA) for total RNA extraction. The ReverTra Ace q-PCRT Master Mix (TOYOBO, Japan) kit was used to reverse transcribe RNA into cDNA, and the SYBR Green Realtime PCR Master kit (TOYOBO, Japan) was used to quantify the mRNA content using the PCR instrument (Stratagene, San Diego, CA, USA, MX3000P) according to the manufacturer’s instructions. miRNA was quantified using the All-in-One TM miRNA Q-PCR Detection System (GeneCopoeia, Rockville, MD, USA). Relative gene expression levels were calculated using the 2^–ΔΔCt method. The primers used were listed as follows: NLRP3 (forward: 5′-CAGACCTCCAAGACCACGACTG-3′; reverse: 5′-CATCCGCAGCCAATGAACAGAG-3′), ASC (forward: 5′-TTATGGAAGAGTCTGGAGCTGTGG-3′; reverse: 5′-AATGAGTGCTTGCCTGTGTTGG-3′), caspase-1 (forward: 5′-TGCCTGGTCTTGTGACTTGGAG-3′; reverse: 5′-ATGTCCTGGGAAGAGGTAGAAACG-3′), β-actin (forward: 5′-GGAGATTACTGCCCTGGCTCCTA-3′; reverse: 5′-GACTCATCGTACTCCTGCTTGCTG-3′), miRNA-33 (5′-TGCGCGTGCATTGTAGTTGCATTGCA-3′). The internal reference was U6.