Sybr qpcr mix

Total RNA was extracted from lung tissues using TRIzol (Vazyme Biotech Co., Nanjing, China), following the manufacturer’s instructions. The isolated RNA was reverse transcribed into cDNA according to the HiScript II Q RT SuperMix protocol for qPCR (Vazyme Biotech Co., Nanjing, China). Real-time PCR was performed using the SYBR qPCR Mix (Vazyme Biotech Co., Nanjing, China). Data were calculated using the 2^-ΔΔt method. Each experiment was performed in triplicate. MasR forward primer 5′-ACAACACGGGCCTCCTATCTG-3′ and reverse primer 5′-GAAGGGCACAGACGAATGCT-3′, β-actin forward primer 5′-GGCTGTATTCCCCTCCATCG-3′ and reverse primer 5′-CCAGTTGGTAACAATGCCATGT-3′.

Total RNA were extracted with an RNA extraction Kit (CWBio, Beijing, China) and quantified. Equivalent amounts of RNA (1 μg) were used for reverse transcription (Vazyme, Nanjing, China). qPCR was performed using SYBR qPCR Mix (Vazyme) and gene specific primers (IL-6, IL-1β, IL-8, TLR2, TLR4 and GAPDH, Table 1) according to manufacturer’s instruction. Expression of target genes were normalized to GAPDH.

Based on the phenotypes of PH and NNMS, we selected extremely high materials R19 (the PH is 118.25 cm, the NNMS is 19.25) and R200 (122.4 cm, 22.6) and extremely small materials R120 (56.2 cm, 15) and R155 (56.4 cm, 15.8) and SN14 (81.0 cm, 18.1). There are significant phenotypic differences at the P = 0.05 level between extremely high line and extremely small lines on PH and NNMS (Supplementary Tables S10 and S11). Extraction of shoot tip tissue from the ternate compound-leaf stage for RNA extraction using TRIzol Reagent (Invitrogen, 15596-026, Carlsbad, CA, USA) was performed. Reverse transcription of extracted RNA into cDNA using the Tianhe Real-time quantitative PCR (RT-qPCR) kit was performed using SYBR qPCR Mix (Vazyme, Q711, Vazyme biotech, Nanjing, China) on the Light Cycler 480 System (Roche, Roche Diagnostics, Basel, Switzerland). The expression levels of candidate genes were calculated with GmUKN1 as an internal reference according to the following formula [65 (link)]:

The qRT-PCR primer sequences specific for candidate genes were designed using Primer Premier 5.0 (http://www.premierbiosoft.com/primerdesign/index.html).

The transcript abundance of all MAPK genes in strawberry was obtained from the transcriptome sequencing data that had been submitted to a public database (NCBI: PRJNA565646, PRJNA552213). The transcript abundance of FaMAPK genes in different development stages and treatments was counted by HTSeq [43 (link)], and FPKM (fragments per kilo base of exon per million fragments mapped) was then calculated to estimate the expression level [44 (link)].
Among all genes, eight FaMAPKs that might be associated with fruit ripening were selected to examine their expression. qRT-PCR was performed with SYBR qPCR Mix (Vazyme Biotech Co., Nanjing, China) using a Bio-Rad CFX96 real-Time System (Bio-Rad, CA, USA). The experiments were repeated in three independent bio-replicates and tech-replicates, and FaActin was used as the internal control to normalize gene expression. The relative expression levels of FaMAPKs were calculated based on the 2^−ΔΔCT method. The primer sequences were designed using Primer Premier 5.0 software (version 5.0; Premier Biosoft International: Palo Alto, CA) and the sequence information is listed in Table S4.

Primers were designed using the Primer-Blast tool at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Sangon Biological Engineering Co., Ltd. (Shanghai, China). The sequences of the primers are listed in Supplementary Table S1. Total cellular RNA was extracted with RNA Trizol (Beyotime Biotechnology). Reverse transcription of RNA was performed using HiScript III RT SuperMix (Vazyme). The SYBR qPCR Mix (Vazyme) was used in a 10-µl reaction mixture that included 1 µl of cDNA template, 0.4 µl of each 0.5 µM primer, 3.6 µl of ddH₂O, and 5 µl of 2× SYBR buffer. The reaction was performed with 1 cycle of 30 s at 95°C and 50 cycles of 10 s at 95°C, 30 s at 60°C, and 15 s at 60°C. Actin was used as the reference mRNA. The qPCR reaction was performed in triplicate.

The total RNA of MLO-Y4 cells was extracted by TRIzol (Biosharp), and cDNA was reverse-transcribed using HiScript 1st Strand cDNA Synthesis Kit (Vazyme) and real-time PCR using SYBR qPCR Mix (Vazyme). The primer sequences were as follows: β-actin (mouse): 5′-CATTGCTGACAGGATGCAGAAGG-3′ (forward) and 5′-TGCTGGAAGGTGGACAGTGAGG-3′ (reverse); IL-6 (mouse): 5′-TACCACTTCACAAGTCGGAGGC-3′ (forward) and 5′-CTGCAAGTGCATCATCGTTGTTC-3′ (reverse); P53 (mouse): 5′-CCTCAGCATCTTATCCGAGTGG-3′ (forward) and 5′-TGGATGGTGGTACAGTCAGAGC-3′ (reverse); P21 (mouse): 5′-TCGCTGTCTTGCACTCTGGTGT-3′ (forward) and 5′-CCAATCTGCGCTTGGAGTGATAG-3′ (reverse); P27 (mouse): 5′-AGCAGTGTCCAGGGATGAGGAA-3′ (forward) and 5′-TTCTTGGGCGTCTGCTCCACAG-3′ (reverse); and Opg (mouse): 5′-CGGAAACAGAGAAGCCACGCAA-3′ (forward) and 5′-CTGTCCACCAAAACACTCAGCC-3′ (reverse).