All in one first strand cdna synthesis supermix

Total RNA was isolated from seeds collected at nine different developmental time points as previously described. Then, using 1 μg of total RNA, cDNA was prepared for RT-qPCR using All-in-one First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China) according to the manufacturer’s instructions. Genomic DNA was removed with the DNase and gDNA Remover included in the commercial kit. PCR primers were designed with Oligo 7.0 software for sequences encoding fatty acid desaturase genes (SAD, FAD2, FAD3, FAD6, FAD7, FAD8) and phospholipids:diacylglycerol acyltransferase genes (PDAT1, PDAT2). All RT-qPCR assays were performed using Top Green qPCR SuperMix (TransGen, China) in a LightCycler 480II Real-Time PCR System (Roche, Mannheim, Germany). The cycling conditions were 95 °C for 15 min, followed by 35 cycles of 95 °C for 10 s and 60 °C for 20 s. Six replicates were analyzed for each target gene. The ubiquitin gene was used as an internal control⁴⁰ . The relative expression levels of target genes were calculated using the 2^-ΔΔCt comparative threshold cycle (Ct) method^{41 (link)}.

Total RNA was isolated with TRIZOL reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The quantity and purity of the obtained total RNA samples were determined by UV spectroscopy (NanoDrop 2000 Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). The sequences of the primers used for the RT-PCR assay are shown in Supplementary Table S1. Two-step RT-PCR was carried out as described in the All-in-One First-Strand cDNA Synthesis SuperMix (AH321-01, TRANSGEN BIOTECH, BeiJing, China) for qPCR protocol. The reverse transcription conditions were as follows: 42 °C for 15 min, followed by 5 s at 85 °C for RT/RI inactivation. qPCR was performed using the following conditions: 30 s at 94 °C for denaturation; 5 s at 94 °C for annealing, and 30 s at 60 °C for extension.

Total RNA was extracted from BV2 microglial cells or ipsilateral brain hemispheres using RNAiso plus (Takara, Kusatsu, Japan). For brain sampling, mice were perfused with autoclaved PBS and their brains were removed. Total RNA (1 µg) was used to generate cDNA by reverse transcription using All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Haidian, China). Then mRNA expression levels of M1 and M2 polarization markers were determined using StepOnePlus^TM qRT-PCR system (Applied Biosystems, Foster city, CA, USA) with FG Power SYBR Green PCR master mix (Life Technologies, Carlsbad, CA, USA) and specific primer sets (Supplementary Table 1). Expression levels of target genes were quantified using the 2^−ΔΔCT method relative to β-actin.

Total RNA was extracted from cells using an EasyPure RNA Kit (TransGen Biotech), and cDNA was synthesized with an All-in-One First-Strand cDNA Synthesis SuperMix (TransGen). The mRNA levels of Usp7 and Usp46 by MPC-11 and MOPC-315 cells were analyzed. Expression was normalized to the expression of the housekeeping gene Gapdh. Primer sets used for these analyses are listed as follows: Gapdh, 5′-AGC TTG TCA TCA ACG GGA AG-3′ (forward) and 5′-TTT GAT GTT AGT GGG GTC TCG-3′ (reverse); Usp7, 5′-AAG TCT CAA GGT TAT AGG GAC GG-3′ (forward) and 5′-CCA TGC TTG TCT GGG TAT AGT GT-3′ (reverse); Usp46, 5′-ATG ACT GTC CGA AAC ATC GCC-3′ (forward) and 5′-TTG ACC AAT CC GAA GTA GTG TTC-3′ (reverse).

Total RNA from cells or tissues was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNAs were generated using All-in-one First-strand cDNA Synthesis SuperMix (Transgene biotech, Beijing, China). qRT-PCR was performed by 7500 Fast Real-Time PCR System (Applied Biosystems, ABI, USA) for 40 cycles using SYBR Green qPCR SuperMix (Transgene biotech, Beijing, China). The relative mRNA expression levels were calculated based on Ct values and normalized to ACTB levels to create internal controls for each sample using the 2^-△△Ct method. Sequences of primers were listed in Supplementary Table S1.

Conidia of D. dactyloides were plated on WA plates covered with cellophane membrane and incubated for 5 days. After treating the resulting cultures with C. elegans from 0 to 24 h, they were collected for RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA). The RNA samples were reverse transcribed using All-in-One First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China) to produce complementary DNA. Real-time PCRs were performed using SYBR PCR mix (TransGen, Beijing, China), and the data were normalized to β-tubulin expression. These experiments were performed in triplicate. The expression fold changes were calculated using the 2^−ΔΔCT method.