Real time pcr detection system

Total RNA from lung tissues was extracted using Trizol according to the manufacturer’s instructions and reverse-transcribed into cDNA using a reverse-transcription kit. qRT-PCR was performed using 2× SYBR Green qPCR ProMix in a real-time PCR detection system (Bio-Rad, USA). The reaction mixture in a 96-well plate was incubated at 95°C for 3 min, followed by 40 cycles at 95°C for 5 s and at 60°C for 30 s. The primer sequences are as follows:
CSRNP1 F:5’-CGAAGGATTGACCGAGAGGAGAAG-3’, R:5’-AGACGCCATCACAGTGACAACC-3’;
JUN F:5’-CGCACCTCCGAGCCAAGAAC-3’, R:5’-GGGTCGGTGTAGTGGTGATGTG-3’;
FAM185A F:5’-AGGTGATGTGGTCTGTCTTGGAAC-3’, R:5’-TGAAGCAAACCATCCTCCGTAGAG-3’.

For the gene expression analysis, total RNA was extracted with the MiniBEST Universal RNA Extraction Kit (TaKaRa, Tokyo, Japan) or the miRNA Mini Kit (QIAGEN, Dusseldorf, Germany) in strict accordance with the instructions of the manufacturers. PrimeScript™ RT reagent (TaKaRa) was used for mRNA reverse transcription, and a miRNA First Strand cDNA Synthesis Tailing Reaction Kit (B532451, Sangon Biotech, Shanghai, China) was utilized for miRNA reverse transcription. After that, the TB Green™ Premix Ex Taq™ II kit (TaKaRa) was utilized to perform qRT‐PCR on a Real‐Time PCR Detection System (Bio‐Rad). U6 was utilized to normalize the miRNA expression levels. For the analysis of miRNA expression in EVs, samples were spiked with cel‐miR‐39 (miRB0000010‐3‐1, RIOBO) to control inter‐sample variability as described in previous studies.¹⁸ For mRNA analysis, β‐actin was employed to normalize the levels of genes of interest. The sequences of all primers used in the present study are shown in Table S1.

Following the 1-week warfarin plus 3-week warfarin-GS regimen, we sacrificed the rats and excised the livers to make liver homogenization (3 mg tissue) with 1 mL TRIZOL reagent (Takara, Japan) and a tissue grinding apparatus. The total RNA was extracted and purified following the instruction by the manufacturer for the preparation of cDNA sample. The liver cytochrome P450 isomer cDNAs were synthesized by reverse transcription reagent (TaKaRa, China). The primers of P450 isomers CYP1A2, CYP2C9 and CYP3A4 were designed as 5′-CGCCCAGAGCGGTTTCTTA-3′ and 5′-TCCCAAGCCGAAGAGCATC-3′, 5′-GGACAGAGACGACAAGCACA-3′ and 5′-CATCTGTGTAGGGCATGTGG-3′, 5′-AAGTCGCCTCGAAGATACACA-3′ and 5′-AAGGAGAGAACACTGCTCGTG-3′, respectively, and applied to analyze the mRNA expression of the isomers during the in vivo warfarin and GS interaction by using the real-time PCR detection system (BioRad, USA).

Total RNA was isolated by using the High Pure RNA Isolation Kit (Roche). The levels of mRNA were determined by reverse transcription quantitative real-time PCR (RT-qPCR). For the detection of human Plk1 mRNA, the 5′-primer sequence was 5′-GAG GAG TAC GGC TGC TGC AAG GAG-3′; the 3′-primer sequence was 5′-GAG ACG GTT GCT GGC CGA GCG TGA-3′. Human β2-microglobulin (B2M) mRNA was used as a control. The 5′-primer sequence of B2M was 5′-TGC TGT CTC CAT GTT TGA TGT ATC T-3′; the 3′-primer sequence of B2M was 5′-TCT CTG CTC CCC ACC TCT AAG T-3′. Briefly, total RNA and primers were mixed with the iTaq Universal SYBR Green One-Step Kit (Bio-Rad) and the mRNA levels were detected using a real-time PCR detection system (Bio-Rad). The expression level of Plk1 mRNA was expressed as the ratio of Plk1 mRNA over B2M mRNA.

Total RNA from HaCaT cells was extracted using the RNeasy Plus Mini kit (Qiagen, Germantown, MD, USA), and cDNA was produced with 500 ng RNA using a reverse transcription kit (ProtoScript^® First Strand cDNA Synthesis Kit, New England Biolabs, Rowley, MA, USA) following manufacturer’s protocol. RNA quantity and quality were measured with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Pre-verified qPCR primers for FGFR1–4 and ADAMs 9, 10, 12, 15, and 17 were purchased from MilliporeSigma (Burlington, MA, USA) (https://www.kicqstart-primers-sigmaaldrich.com, accessed on 17 December 2020). Gene transcripts were measured in triplicates on a real-time PCR detection system (Bio-Rad, Des Plaines, IL, USA) using a PerfeCTa SYBR^® Green FastMix (Quantabio, Beverly MA, USA) and normalized to the reference gene Beta-actin. Thermocycling conditions were set as follows: 1 cycle (95 °C for 5 min), 40 cycles (95 °C for 15 s, 58 °C for 60 s).

Total RNA were extracted using TRIzol reagent (Invitrogen, USA) and reverse-transcribed using the TaqMan Universal Master Mix (Applied Biosystems, USA) or PrimeScript™ RT reagent Kit (TaKaRa, Japan) according to the manufacturer's protocol. The primers used in this study were obtained from Applied Biosystems. The qRT-PCR reaction with SYBR Green (TaKaRa, Japan) was performed using a Real-time PCR Detection System (CFX96, Bio-Rad, USA) in the 25 μl reaction mixtures. The expression levels of the target genes were analyzed using the 2^−ΔΔCt method.

Differential scanning fluorimetry (DSF) was performed as previously described [54 (link)]. A protein/SYPRO orange dye mix containing 4 μM GRASP55 and a 1:5,000 dilution of dye (Life Technologies) were prepared in phosphate-buffered saline (PBS) extemporally. Then, 19.5 μL of the protein/dye mix was aliquoted into a 96-well plate, and 0.5 μL of Graspin (2 mM stock solution in 100% DMSO, 50 μM final concentration) or DMSO control (2.5% final DMSO concentration) was dispensed. The GRASP55/JAM-C DSF experiment was performed by adding JAM-C peptide (1 mM stock in PBS) to the protein/dye mix at a final concentration of 50 μM in the presence of 2.5% DMSO. After sealing with optical tape, thermal melting experiments were performed using a CFX96 (Bio-Rad) Real-time PCR detection system. The plates were first equilibrated at 25°C for 5 min and then heated at increments of 1°C every 60 s, from 20 to 90°C. The fluorescence intensity was recorded at every temperature step using the built-in FRET filter. Raw fluorescence data were evaluated using Microsoft Excel and GraphPad Prism template files adapted from Niesen et al [54 (link)]. After normalization, the melting temperatures (Tm) were measured using a Boltzmann fit equation in GraphPad Prism 5.03.