Revertra ace qpcr rt master mix kit

NRCM (3.0 × 10⁴ cells/well) were incubated at least 24 h before treatment. Total RNA was isolated with a TRI reagent (Sigma, Burligton, MA, USA) from NRCM treated by S protein for 3 h. cDNA was synthesized with a ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed with ABI prism 7500 Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) and KAPA SYBR FAST qPCR kit (Roche, Basel, Switzerland). TNF-α primers were forward 5′- AAATGGGCTCCCTCTCATCAGTTC-3′ and reverse 5′- TCTGCTTGGTGGTTTGCTACGAC-3′ [61 (link)]. IL-1β primers were forward 5′-CACCTCTCAAGCAGAGCACAG-3′ and reverse 5′-GGGTTCCATGGTGAAGTCAA-3′ [61 (link)]. IL-6 primers were forward 5′-TCCTACCCCAACTTCCAATGCTC-3′ and reverse 5′-TTGGATGGTCTTGGTCCTTAGCC-3′ [61 (link)]. ACE2 primers were forward 5′- GCAGATGGCTACAACTATAACCG-3′ and reverse 5′-C CCTCCTCACATAGGCATGAAGA-3′. A total of 18 s rRNA amplification were forward 5′-ATTAATCAAGAACGAAAGTCGCAGGT-3′ and reverse 5′-TTTAAGTTTCAGCTTTGCAACCATACT-3′. Data were normalized with 18 s rRNA.

Total RNA was isolated from GBM cells using the RNA-Quick Purification Kit (Shanghai YiShan Biotechnology; Shanghai, China) according to the manufacturer's protocol. Reverse transcription was conducted using the ReverTra Ace qPCR RT Master Mix Kit (FSQ-101, TOYOBO; Osaka, Japan), and cDNA was used as the template in real-time fluorescence quantification. RT-qPCR was performed with the hot start reaction mix SYBR Green Master (Roche; Basel, Switzerland) on a Real-Time PCR Detection System (Roche 480II). Independent experiments were conducted in triplicate, and ACTB served as an internal control. The following primers were used:
TREM-1: F 5′-TTTGTTTCCCAGTCTGTGTGC-3′, R 5′-TCCCCTATTCTCCATCACCACT-3′; ACTB: F 5′-CATGTACGTTGCTATCCAGGC-3′, R 5′- CTCCTTAATGTCACGCACGAT-3′; CD206: F 5′-CGAAATGGGTTCCTCTCTGGT-3′, R 5′-TTTATCCACAGCCACGTCCC-3′; CD163: F 5′-GTAGTCTGCTCAAGATACACAGAA-3′, R 5′-GCGTTTTGAGCTCCACTCTG-3′; IL1B: F 5′-TGATGGCTTATTACAGTGGA-3′, R 5′-GGTCGGAGATTCGTAGCTGG-3′; CSF1: F 5′-CTCCAGCCAAGATGTGGTGA-3′, R 5′-TCAGAGTCCTCCCAGGTCAA-3′; CSF2: F 5′-AGCCCTGGGAGCATGTGAAT-3′, R 5′-GCAGCAGTGTCTCTACTCAGG-3′; IL6 F 5′-CCTGAACCTTCCAAAGATGGC-3′, R 5′-TTCACCAGGCAAGTCTCCTCA-3′; CXCL: F 5′-TGTGAAGGTGCAGTTTTGCC-3′, R 5′-GGGGTGGAAAGGTTTGGAGT-3′; TGF-α: F 5′-GTTGTAGCAAACCCTCAAGCTG-3′, R 5′-GAGGTACAGGCCTCTGATG-3′; VEGFA: F 5′-AAAACACAGACTCGCGTTGC-3′, R 5′-CCTCGGCTTGTCACATCTGC-3′.

Mouse lung tissues were homogenized using a T10 Basic Ultra-Turrax homogenizer (IKA Labortechnik, Staufen, Germany) and lysed in Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Relative mRNA expression levels were measured by reverse transcription and real-time PCR; 1 µg of total RNA was synthesized to cDNA by reverse-transcription with a ReverTra Ace qPCR RT Master Mix Kit (Toyobo, Osaka, Japan). Real-time PCR was performed in 20 µl reactions containing 10 µl Power SYBR Green^® PCR Master Mix (Applied Biosystems, Waltham, MA), 1 µl cDNA template, 1 µl forward primer, 1 µl reverse primer, and deionized water to bring to the desired volume. Quantitative PCR was performed with the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Waltham, MA) according to the manufacturer’s protocol. Primer pairs for real-time PCR were manufactured by MBiotech (Hanam, South Korea). The levels of mRNA were normalized to IPO8. Fold changes were calculated using the 2^{− Δ ΔCT} method.

Total RNA was isolated using a yeast total RNA extraction kit (Sangon Biotech Co. Shanghai, China). Isolated RNA was treated with RNase-free DNase I (Toyobo, Osaka, Japan) and cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan) as described (Wang et al., 2018 (link)). Real-time PCR was conducted on a Bio-Rad iCycler iQ (Bio-Rad, Hercules, CA, United States) using the THUNDERBIRD SYBR qPCR mix kit (Toyobo, Osaka, Japan). The primers for KmYME and the ACT1 internal control are shown in Additional File 1: Supplementary Table S1. The cycle threshold values (C_T) were determined and the relative fold differences were calculated using the 2^–ΔΔCT method (Nolan et al., 2006 (link)) with ACT1 as the endogenous reference gene. The fold change was shown as the sign of the log₂ transformed fold change (FC) values (log₂ FC).