Aceq universal sybr qpcr master mix kit

To delve deeper into the molecular mechanisms behind capsanthin production, we conducted RNA extraction and RT-qPCR analysis on pepper fruits. Total RNA extraction of pepper fruits was performed according to the instructions of the Polyphenol Total RNA Kit (TIANGEN, Beijing, China, No. DP441), and the cDNA was synthesized using a reverse transcriptase kit (Vazyme, Nanjing, China, No. R323) for RT-qPCR. The ubiquitin gene was used as the reference gene. RT-qPCR analysis was performed using a QuantStudio 1 (ABI) machine and AceQ Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China) to determine the relative expression levels of target genes. Gene-specific primers were designed based on the coding region sequence of each gene using Primer 6.0 software. Three replicates were run for each sample. We quantified the relative changes in gene transcript levels using the 2^−ΔΔCT method [51 (link)]. Table S4 lists all primers used in this study.

Cells were processed to obtain total RNA using Trizol (Thermo Fisher Scientific). The cDNA was then produced through reverse transcription, utilizing the PrimeScript RT Master kit (TAKARA) following the guidelines provided by the manufacturer. The PCR amplification step was carried out with the AceQ Universal SYBR qPCR Master Mix Kit (Vazyme). All quantifications were normalized to the level of the endogenous control GAPDH. The primer sequences used for RT-qPCR analysis are listed in Tables S5.

The qPCR assay was based on the methods reported by Yan et al. [29 (link)]. Total RNA from the mouse spleen was extracted by RNA-easy Isolation Kit (R701, Vazyme, Nanjing, China), and cDNA was synthesized by reverse transcription kit (R312, Vazyme, Nanjing, China) for qPCR assay. AceQ Universal SYBR qPCR Master Mix Kit (Q511, Vazyme, Nanjing, China) was used in the qPCR assay. The total qPCR reaction system was 10 μL, including 5 μL of 2x SYBR Green Master premix (Vazyme, Nanjing, China), 0.2 μL upstream and downstream primers (10 μmol/L), and 1 μL cDNA template. The qPCR reaction process consisted of 40 cycles, including initial denaturation at 95°C for 5 min, denaturation at 95°C for 3 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s. Using GAPDH as an internal reference, the formula 2^−ΔΔCt was used to calculate the relative gene expression. The PCR primer sequence is shown in Table 2, which was synthesized by Suzhou GENEWIZ Company (Suzhou, China).

Total RNA was separated from HCC cell lines with an RNA isolater (Vazyme, #R401-01-AA), and its concentration was determined using NanoDrop ONE (Thermo Fisher Scientific). The RNA was then reverse-transcribed into cDNA using a reverse transcription kit (Vazyme, #R323-01). qRT-PCR detection was performed using the AceQ Universal SYBR qPCR Master Mix kit (Vazyme, #Q511-02) in the QuantStudio™ 5 Real-Time PCR (Thermo Fisher Scientific) (PRC1: forward primer: CGC​CAT​GAG​GAG​AAG​TGA​GG, reverse primer: TTG​TAA​CCG​CTG​GTC​CTC​TG; RACGAP1: forward primer: ACG​TTG​AAT​AGG​ATG​AGT​CAT​GGA, reverse primer: AAA​GTC​CTT​CGC​CAA​CTG​GA). All procedures were conducted in accordance with the manufacturer’s instructions. The thermal cycling parameters for amplification were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 10 s, and annealing and extension at 60°C for 1 min for a total of 40 cycles.

Total RNA of P. trichocarpa was extracted using the CTAB (cetyltrimethylammonium bromide) method (Jaakola et al., 2001 (link)), and RNA samples were analyzed by agarose gel electrophoresis to ensure integrity. HiScript® II Q Select RT SuperMix for qPCR Kit (Vazyme) was used for reverse transcription, and gDNA Wiper Mix to remove the genome DNA. qRT-PCR was performed using the AceQ® Universal SYBR qPCR Master Mix Kit (Vazyme) following the manufacturer’s instructions. The qRT-PCR was performed on a qTOWER 3G Cycler (Analytik Jena, Germany), and the 2^−ΔΔCT method was used to analyze the relative gene expression level. Primer Premier 5 was used to design the primers, and NCBI primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) was employed to check their specificity. The P. trichocarpa actin gene (GenBank ID: XM_002298674) was used as the reference gene (Chu et al., 2014 (link)). All PtrAHL gene-specific primers used for qRT-PCR are listed in Table S1.

Fibroblasts were treated with IL-1β (10 ng/mL) and IPFSC-EVs (10⁹ particles/mL) for 24 h, and total RNA was extracted using TRIzol (Tiangen, Beijing, China), after which cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing, China). Subsequently, RT–PCR was performed using the AceQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). Gene expression levels were determined using the 2^−∆∆CT method. The primer sequences used for RT–PCR are listed in Table 1.

Total RNA content was extracted from the tissues and cells using the TRIzol agent (16096020, Thermo Fisher Scientific). The cDNA of miRNA was synthesized using the miRNA First Strand cDNA Synthesis kit (Tailing Reaction) (B532451, Sangon Biotech), while the mRNA was obtained using the BeyoRT^TM II First Strand cDNA Synthesis kit (RNase H-) (D7168L, Beyotime, Shanghai, China). Subsequently, RT-qPCR was performed using the AceQ^® Universal SYBR qPCR Master Mix kit (Q511-02, Vazyme Biotech, Nanjing, China) on a CFX96^TM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, United States). U6 was regarded as the internal control for miR-324-3p, while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control for lncRNA 74.1 and NRG1. The primer of U6 was obtained using the microRNA reverse transcription kit, and the primer sequences of lncRNA 74.1, miR-324-3p, NRG1, and GAPDH were provided by Sangon Biotech (Supplementary Table 1). Fold changes were calculated based on the 2^–ΔΔCt method.