Venous blood samples were obtained from untreated, active and hospitalized AOSD patients who had available samples at the time of inclusion (N = 20) and healthy controls (N = 21). Briefly, PBMC was isolated by density gradient centrifugation on Lymphoprep (Axis-Shield, Dundee, UK) according to the manufacturer's instructions (19 (link)). The collected cells were used for RNA extraction using RNAeasy columns (Qiagen, Hilden, Germany), and cDNA was prepared by PrimeScript RT Master Mix transcription kit (TaKaRa, Tokyo, Japan). The mRNA expression levels of DNA-sensors expression were determined by SYBR green master mix (Takara). To standardize mRNA expression levels of DNA-sensors, the mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were also determined in parallel for each sample. Relative mRNA expression was calculated as described previously.
Primescript rt master mix transcription kit
Manufactured by Takara Bio
Sourced in Japan
The PrimeScript RT Master Mix transcription kit is a reagent used for reverse transcription of RNA into cDNA. It contains all the necessary components for this process in a single, convenient-to-use solution.
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Lab products found in correlation
4 protocols using primescript rt master mix transcription kit
1
AOSD Patients' DNA-Sensor mRNA Expression
2
Quantification of Mitochondrial DNA Levels
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mRNA was isolated and cDNA was prepared (PrimeScript RT Master Mix transcription kit, TaKaRa, Tokyo, Japan). RT-PCR was performed to detect mitochondrial and chromosomal genes. The RT-PCR contained 4 ng of DNA, isolated as described above, SYBR green master mix (Takara), and primers for cytochrome B, cytochrome C oxidase subunit III, and GAPDH. The enzyme was activated at 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The results are expressed as mtDNA copy numbers [24 (link), 25 (link)].
3
Quantification of Inflammatory Cytokines in Liver
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Total RNA was extracted from liver tissues with TRIzol Reagent (15596026, Thermo Fisher Scientific, California, United States) according to the manufacturer’s specifications. Reverse transcription was completed by a Prime-Script RT Master Mix transcription kit (RR036A, TaKaRa, Tokyo, Japan). mRNA-specific primers (Sangon Biotech, China) were used to detect the RNA expression of IL-6, IL-1β and TNF-α. A 7500 Fast system (Applied Biosystems, United States) was used for qRT-PCR analysis. The results were analyzed using the 2−ΔΔCt method, and GAPDH was amplified as an internal standard. The primer sequences are listed in Supplementary Table S1 .
4
RT-PCR Analysis of Inflammatory Genes
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mRNA was isolated and cDNA was prepared (Prime-Script RT Master Mix transcription kit, TaKaRa, Tokyo, Japan). RT-PCR was performed to detect NETs and LPS-stimulated MH-S macrophage genes. The RT-PCR contained 4 ng of DNA, isolated as described above, SYBR green master mix (Takara), and primers for IL-1β, IL-6, TNF-α, and β-actin. The enzyme was activated at 95°C for 30 s, followed by 40 cycles at 95°C for 15 s, and 60°C for 60 s. Primer sequences were designed as follows: 5′-TCGCAGCAGCACATCAACAAGAG-3′ (forward) and 5′-TGCTCATGTCCTCATCCTGGAAGG-3′ (reverse) for mouse IL-1β; 5′-TGGGACTGATGCTGGTGACA-3′ (forward) and 5′-ACAGGTCTGTTGGGAGTGGT-3′ (reverse) for mouse IL-6; 5′-ATGTCTCAGCCTCTTCTCATTC-3′ (forward) and 5′-GCTTGTCACTCGAATTTTGAGA-3′ (reverse) for mouse TNF-α; 5′-CTACCTCATGAAGATCCTGACC-3′ (forward) and 5′-CACAGCTTCTCTTTGATGTCAC-3′ (reverse) for mouse β-actin.
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