Aceq universal sybr qpcr master mix

Total cellular RNA was isolated from HMC3, U87, HS683, U251, and U118 cells using the TRIZOL reagent (15596-018, Invitrogen, USA), dissolved in RNase-free H₂O, and stored at -80°C. cDNA synthesis was performed using M-MLV Reverse Transcriptase (AT341, TransGen, China) following the manufacturer’s instructions. The PCR system was used to amplify all transcripts using the AceQTM Universal SYBR Qpcr Master Mix (Q511-03, vazyme). A reaction mixture (total volume, 20 µL) was prepared using cDNA (8 μL), SYBR^® Green Master Mix (10 μL), upstream and downstream primers (0.5 μL), and ddH₂O (1 μL). The qPCR was performed under the following conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, and annealing at 60°C for 30 s. Steps 2–3 were repeated for 40 cycles, and thereafter at 95°C for 15 s, at 60°C for 60 s, at 95°C for 15 s, and at 4°C. Target gene mRNA expression was verified and analyzed using the Illumina eco real-time PCR system using GAPDH as an internal standard. Quantification was performed by normalizing the expression levels of the target genes to the GAPDH expression levels; the expression levels of the genes were calculated using the 2^-ΔΔCt method. All experiments were performed in triplicate.

The total RNA of BMDMs was extracted using TRIzol reagent (Thermofisher) and performed reverse transcription with 5x HiScript II Q RT SuperMix (Vazyme). Real-time qPCR amplification of reverse transcription products was performed using 2x AceQ Universal SYBR qPCR Master Mix (Vazyme). The primers of each cytokine and gene are displayed in Table 1.
Table 1

Primers for qPCR

Gene	Forward Primer (5’-3’)	Reverse Primer (5’-3’)
Tnf-α	CCCTCACACTCAGATCATCTTCT		GCTACGACGTGGGCTACAG
Il-1β	GCAACTGTTCCTGAACTCAACT		ATCTTTTGGGGTCCGTCAACT
Nos2	GTTCTCAGCCCAACAATACAAGA		GTGGACGGGTCGATGTCAC
Il10	GCTCTTACTGACTGGCATGAG		CGCAGCTCTAGGAGCATGTG
Arg1	CTCCAAGCCAAAGTCCTTAGAG		AGGAGCTGTCATTAGGGACATC
Prmt1	CTTGGCTAATGGGATGAGCCT		GCGTTGGGCTTCTCACTACTT
Prmt2	GGAAGACCCCGTGGACTATG		CCGTGGTTTGTCTCAGGATAAGA
Prmt3	CAGAGCACCAAAACACACTGG		TCAGGGTCACAATGAGGGAAC
prmt4	TGACATCAGTATTGTGGCACAG		CTGAGGAGCCTAAGGGAATCA
Prmt5	CTGAATTGCGTCCCCGAAATA		AGGTTCCTGAATGAACTCCCT
Prmt6	ATGTCGCTGAGCAAGAAAAGA		CGGAGTAGCACTCGTAGTACAG
Prmt7	GCCAGGTCATCCTATGCCG		GCCAATGTCAAGAACCAAGGC
Prmt8	ACGTGGTAGCAATCGAAGACA		GCTCCTTCATGGCAACATCC
Prmt9	CCTGAAAGAGTGTCCCTAGTTGT		TGCAGAAGTAAATGTTCCCATGC

DMEM was purchased from BasalMedia. Penicillin/streptomycin and FBS were acquired from Gibco. TRIzol reagent was obtained from Thermofisher. 5x HiScript II Q RT SuperMix and 2x AceQ Universal SYBR qPCR Master Mix were purchased from Vazyme. Recombinant murine M-CSF, IFN-γ, and IL-4 protein were purchased from Peprotech. The antibodies are listed as follows: p-STAT1 (Abclonal), STAT1 (Abclonal), p-STAT3 (Abclonal), STAT3 (Cell Signaling Technology, CST), p-STAT6 (Abcam), STAT6 (Abclonal), p-ERK (CST), ERK (CST), p-JNK (CST), JNK (CST), p-P65 (CST), P65 (CST), p-P38 (CST), P38 (Abclonal), iNOS (CST), Arg1 (CST), PRMT2 (Novus), β-actin (CST), PE anti-mouse F4/80 (Biolegend), FITC anti-mouse CD86 (Biolegend), APC anti-mouse CD206 (Biolegend).

Total RNA was purified from frozen hearts using TRIzol reagent (Invitrogen), and 500 ng of RNA was reverse transcribed to cDNA using a 5×HiScript II Q RT SuperMix for qPCR (Vazyme, China). qRT-PCRs were performed using a 2×AceQ Universal SYBR qPCR Master Mix (Vazyme, China) according to the manufacturer's protocols. Fold change of gene expression was calculated using 2^-ΔΔCt method after normalized to a housekeeping gene (18S rRNA). Primer sequences are as follows:
NPNT-Forward, 5'-GAAGCCTCGGCCCTGTAAG-3', NPNT-Reverse, 5'-AGCATGTATCCGTTGAGACAGTA-3', 18S-Forward, 5'-AGTCCCTGCCCTTTGTACACA-3', 18S-Reverse, 5'-CGTTCCGAGGGCCTCACT-3'.

Total RNA was extracted using the Eastep® Super Total RNA Extraction Kit (Promega, Beijing, China) and reverse transcribed into complementary DNA (cDNA) by a HiScript III 1st Strand cDNA Synthesis Kit with gDNA wiper (Vazyme) with Oligo (dT)₂₀ and random hexamers. RT–qPCR was performed on a CFX Connect qPCR System in a 20 μL volume system containing 4 μL of 5 μM of each primer, 10-fold-diluted cDNA, and 10 μL 2 × AceQ® Universal SYBR qPCR Master Mix (Vazyme). Arabidopsis ACTIN II (AT3G18780) and GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPC2; At1g13440) and soybean ACTIN 7 (GmACT7; LOC100806695) and CONSTITUTIVE 4 (GmCONS4; LOC100783869) were used as the internal controls. All primers were listed in Supplementary Table 1.

The RNA was extracted from samples of six developmental stages of plum using the Tiangen kit and was reverse transcribed to cDNA using the Vazyme Reverse Transcription Kit (Vazyme, Nanjing, China, code: R212). The RT-qPCR analysis was performed on an ABI QuantStudio 1 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR green (Vazyme Biotechnology, Nanjing, China) in a 10 μL reaction mixture containing 1 μL diluted cDNA, 10 μM of each primer 0.4 μL, ddH₂O 3.2 μL, and 5 μL 2 × AceQ Universal SYBR qPCR Master Mix. The PCR reaction protocol was 95 °C for 2 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. The qRT-PCR results were calculated using the 2^−ΔΔCT method. Three biological replicates were established for each sample. The CAC and Ubi genes were used as housekeeping genes in Prunus salicina and pepper, respectively [2 (link)]. All the primers used in the PCR analysis are listed in (Table S1).

Total RNA was extracted from the white muscles and cells following the protocols of Trizol reagent (Vazyme, Nanjing, China), and dissolved in DEPC-treated water. Then calculated quantity and purity of them according to the method of [40 (link)]. Following the manufacturer's instructions, cDNA was generated from 500 ng DNase-treated RNA by a qPCR Kit (Vazyme, Nanjing, China), then diluted with DEPC-treated Water and stored at -20°C until use. The endogenous reference gene was ef1α (elongation factor 1 alpha). Designed Gene primers from available sequences using Primer 5 software (Table 2).
Real-time qPCR assays were performed with a real-time detector (BIO-RAD, USA). The assays were performed with a reaction mix of 20 μL per sample, each of which contained 2 μL template (100 ng cDNA), 10 μL 2 × AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 0.4 μL of each primer (10 μmol L⁻¹) and 7.2 μL dH₂O. The procedure was set as follows: initial denaturation at 95°C for 5 min followed by 40 cycles, annealing at 95°C for 10 s and a final extension at 60°C for 30 s, followed by a melt curve analysis of 15 s from 95 to 60°C, 1 min for 60°C, and then up to 95°C for 15 s. The expressions of target gene were calculated by using the 2^−ΔΔCT method [41 (link)] with ef1α as the calibrator.