Real time pcr detection system

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′.

Total RNA was isolated using the RNeasy Mini kit (Qiagen, 74104) according to the manufacturer’s protocol. A cDNA sample, prepared from 1 μg total RNA, was used for quantitative reverse transcription polymerase chain reaction (RT-PCR) performed with the High Capacity cDNA Reverse Transcription Kit (Life Technologies, 4368814) or iScript Reverse Transcription Supermix (BIO-RAD, 1708840). Quantitative PCR (qPCR) was done with the Power SYBR Green Master Mix (Life Technologies; 4368708); data were collected and analyzed on a Bio-Rad Real-Time PCR Detection System or a Roche LightCycler 480 qPCR instrument. Thermal-cycling parameters for the PCR were as follows: 95°C for 10 min, followed by 45 cycles each of 95°C for 20 s, 60°C for 60 s. The relative quantity of mRNA was normalized against the relative quantity of RPLP0 or GAPDH mRNA in the same sample. Primer sequences in a 5′ to 3′ orientation are shown in Supplementary file 1.

Total RNA was extracted using RNAiso Plus (Takara, Japan) according to the manufacturer’s instructions. The PrimerScript^{^TM} RT Reagent Kit with gDNA Eraser (Takara, Japan) was used to synthesize first-strand cDNA from equivalent amounts of RNA. Then, quantitative real-time PCR was performed using SYBR Premix EX Taq^{^TM} (Takara, Japan) following a real-time PCR detection system (Roche, CH). Relative gene expression was normalized to the housekeeping gene 36B4 using the –ΔΔCt method. Primer sequences are summarized in Supplementary Table 2.

Total RNA of cell lysates was extracted in accordance with TRIzol solution (Invitrogen Life Technologies, Carlsbad, CA, USA). 2 μg of RNA was taken to synthesize cDNA using M-MLV Reversereverse transcription reaction system (Invitrogen). oligo (deoxythymidine)₂₀ was used as primers.
Quantitative real-time PCR was conducted using BioRad Real-Time PCR detection system, using SYBR (Roche). Relative expression levels were determined using 2^−ΔΔCt methods. GAPDH was applied as the internal control and amplified. The amplification protocol included denaturation at 95°C for 1 min, annealing at 60°C for 10 sec, extension at 72°C for 30 sec for 35 cycles. All the experiments were performed for three times.

Total RNA was extracted from cells with TRIzol reagent (Takara, T9108) according to the manufacturer’s instructions. cDNA was synthesized using 800 ng of total RNA with a cDNA reverse transcription kit (CWBID, CW0741S). SYBR Premix Ex Taq™ (CWBIO, CW2602M) and a Roche real-time PCR detection system were used to quantify circRNA. The primer sequences for circNFIB were as follows: forward, 5’-tgaacgagatcaagcaccat-3’ and reverse, 5’-ctgctcggtgacag-3’. GAPDH was used as an internal control, and the primer sequences were as follows: forward, 5’-agccatgtacgtagccatcc-3’ and reverse: 5’-ctctcagcttggtggtgaa-3’.