7300plus real time pcr system

Representative DEGs were additionally verified using qRT-PCR using a 7300 Plus Real-Time PCR System (Thermo Fisher, Pittsburg, PA, USA). Total RNA was reverse-transcribed into cDNA using an All-in-One RT Master Mix and qPCR reactions utilized Eva Green qPCR Master Mix (Applied Biological Materials, Vancouver, Canada) with the following cycles: 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 63°C for 30 s. The relative expression level of target genes was measured with the 2^-ΔΔCt method (Kenneth et al., 2001 (link)) and 16S rRNA was used as the reference gene. All tests were performed in triplicate using primers listed in Table 1.

TRIzol reagent (Sigma‐Aldrich) was used to extract total RNA from the midbrain. Afterward, RNA was reverse transcribed to cDNA using a PrimeScript RT reagent kit (Takara). Real‐time PCR was conducted using the SYBR Premix EX Taq I (Takara) on a 7300 Plus Real‐time PCR System (Thermo Fisher). Each reaction mixture contained 1 μL of cDNA template, 5 pmol primer, 10 μL PCR master mixture, and water. The thermocycling conditions used were an initial 95°C for 15 s, followed by 40 cycles at 94°C for 10 s and 60°C for 25 s. The relative mRNA levels of genes analyzed were obtained using GAPDH mRNA as the internal standard. The comparative threshold cycle method was used to analyze the data. The primer sequences are shown in Table 1.

Total RNA was obtained from cultured bladder tumor cells, CAFs, and human tissues with a total RNA extraction kit of UNIQ-10 column Trizol type (Sangon Biotech, China) following the instruction of the manufacturer. Reverse transcription was subsequently performed utilizing the RR047 cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA) and qRT-PCR was performed in a 7300 Plus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) using the Phusion U Green Multiplex PCR Master Mix (Invitrogen, United States). The mRNA expression of genes was normalized to the levels of GAPDH expression. The sequences of primers were as follows: JUNB-F:5′-ACAGTACTTTTACCCCCGCG-3′, JUNB-R: 5′-TGA​GCG​TCT​TCA​CCT​TGT​CC-3’.

Trizol (Invitrogen, USA) was used for segregating total RNA followed by reverse transcription of lncRNA PART1 and SOCS6 with BeyoRT™II Kit (Beyotime, Shanghai, China). Reverse transcription of miR-17a-5p was conducted using Mir-X Kit (Takara, Japan). Then, 7300Plus Real-Time PCR System (Applied Biosystems, USA) with SYBR Green (Applied Biosystem) was applied for RT-qPCR: predenaturation, 95 °C, 30 s and (denaturation, 95 °C, 5 s, annealing, 60 °C, 10 s and extension, 72 °C, 30 s) × 35 cycles. Primers were listed as below, which were: lncRNA PART1 5′-CAATAAGGCAGAAGAAGGTG-3′ (forward), 5′-GGAGAATCTGAAGTCCCAAG-3′ (reverse)[18 (link)]; miR-17-5p 5′-CGGCGGCAAAGTGCTTACAG-3′ (forward), 5′-GTGCAGGGTCCGAGGT-3′ (reverse)[19 (link)]; SOCS6 5′-GGAATTCATGAAGAAAATCAGTCTGAA-3′ (forward), 5′-CGGAATTCTCAGTAGTGCTTCTCCTGCA-3′ (reverse)[20 (link)]; GAPDH 5′-CCTGCCTCTACTGGCGCTGC-3′ (forward), 5′-GCAGTGGGGACACGGAAGGC-3′ (reverse) and U6 5′-CTCGCTTCGGCAGCACA-3′ (forward), 5′-AACGCTTCACGAATTTGCGT-3′ (reverse). GAPDH and U6 were used for normalization of lncRNA PART1, SOCS6, and miR-17-5p, respectively and the relative expression levels were analyzed using 2^-ΔΔCt method.

RNA from the mouse corpus callosum tissues and cells was extracted using the Qiagen RNeasy kit (74106, Qiagen, Germany) according to the manufacturer protocol and the concentration of RNA was measured by nanodrop (Thermo Fisher, USA). After that, cDNA was prepared using the PrimeScript RT Master Mix Reverse Transcription Kit (RR036, Takara, Japan) according to the manufacturer protocol. Gene expression was determined by qRT-PCR on 7300Plus Real-Time PCR System (Applied Biosystems, USA) using TB Green Premix Ex Taq (RR420, Takara, Japan). Relative gene expression was normalized to the expression of housekeeping gene GAPDH. Primers used are listed below.
Mouse: Gapdh (Forward 5′GACCCCTTCATTGACCTCAAC3′, Reverse 5′GATGACCTTGCCCACAGCCTT3′),
Ahr (Forward 5′AGGACCAGTGTAGAGCACAAA3′, Reverse 5′AAGCATAGAAGACCAAGGCA3′),
Arnt (Forward 5′ATCGGTCTAGTTCCAGTGAGC3′, Reverse 5’TGTAGGTGTTGCTTTGGTCTC3′),
Cyp1a1 (Forward 5′TCTTCAGGCTTAGACTGTCCA3′, Reverse 5’CAGGATCTGTGTTTCTGGCTA3′),
Syk (Forward 5′GGCTCACAACAGGAAGGCACAC3′, Reverse 5′TGGGAGTGGTAATGGCAGAGGTC′),
Nos2 (Forward 5′TCCAGAATCCCTGGACAAGCTGC3′, Reverse 5′TGCAAGTGAAATCCGATGTGGCCT3′),
Il-1b (Forward 5′TACATCAGCACCTCACAAGC3′, Reverse 5′AGAAACAGTCCAGCCCATACT3′),
Il-6 (Forward 5′CTTCTTGGGACTGATGCTGGTGAC3′, Reverse 5′TCTGTTGGGAGTGGTATCCTCTGTG3′),
Ccl2 (Forward 5′CACTCACCTGCTGCTACTCATTCAC3′, Reverse 5′CTTCTTTGGGACACCTGCTGCTG3′).

Total RNA was extracted from the RAW 264.7 mouse macrophage cell with the use of an RNeasy Fibrous Tissue Mini-Kit (Qiagen, Hilden, Germany) and subjected to reverse transcription (Wang et al., 2019 (link)). The resulting cDNA was subjected to a quantitative real-time polymerase reaction chain (RT-PCR) analysis with primers specific for CTSS, p22^phox, p47^phox, gp91^phox with the use of an 7300 Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The transcription of targeted RNAs was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. The primer sequences are listed in Table 4.

MDMs derived from 3 healthy individuals or mTHP-1 cells were treated with THZ1 for 0.5–1 h before or after stimulation. Total RNA was extracted from cell lysates and reverse transcribed to cDNA using a reverse-transcriptional kit (TaKaRa). RT-PCR was performed in a triplicate with the SYBR Prime Script RT-PCR kit (TaKaRa) on the 7300 plus Real-Time PCR system (Applied Biosystems). The primer sequences are described in the Supplementary Materials and Methods.