Sybr green 1

After extracting total RNA from microglial cell line BV2 with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), complementary DNA was synthesized from 2 μg of total RNA. Quantitative polymerase chain reaction (qPCR) was performed with SYBR Green I (GeNet Bio, Nonsan, Korea) in a LightCycler 480 system (Roche, Rotkreuz, Switzerland). Relative expression of mRNA was measured using comparative CT (ΔΔCT), normalized to GAPDH, and described as the fold change over control. Primers are as follows [28 (link)]; GAPDH, Forward (5′-3′) GTG-GCA-AAG-TGG-AGA-TTG-TTG, Reverse (5′-3′) CGT-TGA-ATT-TGC-CGT-GAG-TG; TNFα, Forward (5′-3′) TGG-GAC-AGT-GAC-CTG-GAC-TGT, Reverse (5′-3′) TTC-GGA-AAG-CCC-ATT-TGA-GT; IL-1β, Forward (5′-3′) AAG-GGC-TGC-TTC-CAA-ACC-TTT-GAC, Reverse (5′-3′) ATA-CTG-CCT-GCC-TGA-AGC-TCT-TGT; IL-6, Forward (5′-3′) ATC-CAG-TTG-CCT-TCT-TGG-GAC-TGA, Reverse (5′-3′) TAA-GCC-TCC-GAC-TTG-TGA-AGT-GGT; iNOS, Forward (5′-3′) GCT-CAT-GCG-GCC-TCC-TTT, Reverse (5′-3′) CCT-GGT-ACG-GGC-ATT-GCT; COX-2, Forward (5′-3′) TGC-TGT-ACA-AGC-AGT-GGC-AA, Reverse (5′-3′) AGG-GCT-TTC-AAT-TCT-GCA-GCC-A.

Wild-type plants (cv. Dongjin) and lg1 mutants were grown under growth chamber conditions prior to RNA extraction. Subsequently, 1 cm of the collar region was collected and immediately frozen in liquid nitrogen. Total RNA was isolated manually (RNAiso; Takara Bio, Shiga, Japan) and quantified using a NanoDrop Spectrophotometer ND-2000 [82 (link)]. After synthesizing cDNAs using SuPrimeScript RT Premix [with oligo (dT), 2×] (GeNet Bio, Daegu, Republic of Korea), qRT-PCR was performed on a Rotor Gene Q instrument system (Qiagen, Hidden, Germany) using SYBR Green I (GeNet Bio, Republic of Korea). All reactions were conducted in three biological replications, and data analysis was performed using the 2^−ΔΔCt method, as previously described [83 (link)]. For other tissues, samples were collected as previously reported [84 (link)]. All the primers used in this study are listed in Table S4.

Total RNA was extracted from the leaves, roots, and Oc cells using RNAiso Plus (Takara, Japan). After combining 3 µg of RNA with 10 ng of oligo (dT), the first-strand cDNA was synthesized using 100 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA), 100 units of RNasin (Promega), and 2.5 mM deoxyribonucleotide triphosphates. The gene expression levels were analyzed by quantitative RT-PCR using SYBR Green I, Prime Q-Matermix (2x) (GENET Bio, Daejeon, Korea) in a Rotor-Gene Q system (Qiagen, Hilden, Germany). Rice Actin1 (OsActin1) was used as an endogenous control to normalize the expression level of the respective genes. All experiments were repeated at least three times, and the relative transcript levels were calculated using the ΔΔCt method (Schmittgen and Livak, 2008 (link); Yoon et al., 2022 (link)). The primers used in this study are listed in Supplementary Table 1.