Revertra ace qrt pcr kit

Total RNA was extracted with TRIzol reagent (Tiangen, China) according to the manufacturer's protocol. The mRNA was then transcribed into cDNA using ReverTra Ace® qRT-PCR Kit (Toyobo, Japan). The cDNA abundance was measured by quantitative real-time PCR (qRT-PCR) using SYBR® Green Real-Time PCR Master Mix on CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA). Then, qRT-PCR was performed to quantitate the mRNA of Nlrp3, IL-1β, IL-18, and actin. The sequences of primers used for PCR were as follows: 5′-ATCAACAGGCGAGACCTCTG-3′ and 5′-GTCCTCCTGGCATACCATAGA-3′ for Nlrp3; 5′-GAAATGCCACCTTTTGACAGTG-3′ and 5′-TGGATGCTCTCATCAGGACAG-3′ for IL-1β; 5′-GACTCTTGCGTCAACTTCAAGG-3′ and 5′-CAGGCTGTCTTTTGTCAACGA-3′ for IL-18; 5′-ACAAGGCACGGGACCTATG-3′ and 5′-TCCCAGTCAGTCCTGGAAATG-3′ for caspase-1; 5′-GGGAAATCGTGCGTGACATCAAAG-3′ and 5′-CATACCCAAGAAGGAAGGCTGGAA-3′ for β-actin. Primer specificity was determined by melt curve analysis. Actin was used as the endogenous control to estimate other genes. The results are presented as fold induction over levels in untreated control cells. Relative gene levels were calculated according to the comparative Ct method formula 2^−ΔΔCt.

The TRIzol (Invitrogen, Carlsbad, CA, USA) was employed to extract total RNA. ReverTra Ace qRT-PCR kit (Toyobo, Osaka, Japan) was used for RNA reverse transcription to acquire cDNA. qRT-PCR was performed on the products with THUNDERBIRD SYBR® qPCR Mix (Toyobo, Osaka, Japan). PCR procedures were predenaturation at 94°C for 2 min, denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 1 min, repeat the previous procedure for 30 times, and continuous extension at 72°C for 10 min. U6 and β-actin were selected as internal references for miR-30a-5p and ANLN, respectively. Results were shown by 2^-ΔΔCt value. Primer sequences are presented in Table 1.

To synthesis cDNA, total RNA from diluted stocks of the same RNA that was subjected to RNA-seq was used in each reverse transcription reaction using the ReverTra Ace qRT-PCR Kit (Toyobo, Japan). qRT-PCR was performed using SYBR-Green chemistry and the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) [32 (link)]. The primers used to amplify the targeted genes were designed using Primer Premier 5.0 (Additional file 3: Table S2). Melting curve analysis of the PCR products was conducted at the end of each PCR cycle to verify the amplicon specificity. The mRNA expression levels of the target genes were normalized relative to the expression of the housekeeping gene actin2 to minimise sample variation. All qRT-PCR reactions were repeated with three independent biological replicates and two technical replicates. The data were analysed based on the method of Livak and Schmittgen [33 (link)].

Total RNA was extracted from the prefrontal cortex and hippocampus using TRIzol (Invitrogen) according to the manufacturer’s instructions. 1 µg RNA was reverse-transcribed into cDNA using the ReverTra Ace qRT-PCR kit (Toyobo). Real-time PCR was performed using the SYBR Premix MASTER Kit (Roche) with a LightCycler 480 Instrument (Roche). All data for each sample were collected in triplicate. Standard curves were generated, and the relative amount of miRNA was normalized to the amount of U6 (2^−ΔΔCt).

Total RNA was isolated using RNAiso Plus (9108, Takara). Total RNA (0.5–1 μg) was reverse-transcribed with a ReverTraAce qRT-PCR kit (FSQ-101, Toyobo) according to the manufacturer's protocol to synthesize cDNA. Real-time PCR was performed using a SYBR Premix Ex TaqTM Kit (QPK-201, Toyobo) with specific primers. The reactions were performed by using a Lightcycler 2.0 instrument (Roche Applied Science). The mRNA levels were normalized to GAPDH. All data for each sample were collected in triplicate. The fold changes were calculated by relative quantification (2^−ΔΔCt). The specific primers used in the present study are listed in Supplementary Table 1.

Q-PCR was conducted to measure the expression levels of U251 and U87 cells. Total RNA was isolated using RNAiso Plus (9108, Takara). Total RNA (0.5–1 μg) was reverse-transcribed with miR224-3p, miR210 stem-loop and U6 snoRNA RT primers (RiboBio, Guangzhou, China) using a ReverTraAce qRT-PCR kit (FSQ-101, Toyobo) according to the manufacturer's protocol to synthesize cDNA. Real-time PCR was performed using a SYBR Premix Ex TaqTM Kit (QPK-201, Toyobo) with miR224-3p, miR210 and U6 snoRNA primers (miRQ0009198-1-1 for miR224-3p, miRQ0000267-1-1 for miR210, MQP-0201 for U6, RiboBio) as previously described [21 (link), 52 (link)]. The reactions were performed using a Lightcycler 2.0 instrument (Roche Applied Science). mRNA levels were normalized to GAPDH. U6 expression was used as the endogenous control for miRNA level. All data for each sample were collected in triplicate. The fold changes were calculated by relative quantification (2^−ΔΔCt). The primers used in the present study are listed in Supplementary Table S1.

Total RNA was extracted from cells or frozen mouse tissues using a TRIzol reagent (Invitrogen). cDNA samples were produced using the ReverTra Ace qRT-PCR Kit (TOYOBO). qPCR-RT was performed by the SYBR green method using a Thunderbird qPCR Mix (TOYOBO) and a StepOne Plus Sequence Detector (Applied Biosciences). Data are presented as the mean ± standard deviation (SD). Fold differences among groups were calculated by the ΔΔCt method. 36B4 (Rplp0) gene was used as an internal control. The primers are listed in Supplementary file 3.