Nanodrop 2.0 spectrophotometer

RAW264.7 cells (2 × 10⁵ cells) were seeded on a 60-mm culture dish for 2 h in a CO₂ incubator. Then, cells were applied to a differentiation medium and treated with 300 µM of melatonin for 1, 3, and 5 days. Total RNA and cDNA were prepared using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The total RNA concentration was determined with a NanoDrop 2.0 spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA). A quantitative real-time PCR was performed with Maxima™ SYBR Green/ROX qPCR Master Mix (Fermentas) and run on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems 7500 System, Foster City, CA, USA) using Sequence Detection System software version 2.0.1. The relative mRNA levels were normalized using GAPDH as a housekeeping gene. Atp6v0d2 (NM_152565) and GAPDH (NM_008084) messenger RNA expressions were quantified through QuantiTect Primer Assays (Qiagen, Hilden, Germany).

Total DNA from soil was extracted using the PowerSoil^® DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, United States) according to the manufacturer’s instructions. The extracted DNA was examined for quantity and quality using a Nanodrop 2.0 spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, United States). The paired primers ITS3-F (5′-GCATCGATGAAGAACGCAGC-3′) and ITS4-R (5′-TCCTCCGCTTATTGATATGC-3′) were used to amplify the ITS2 region in the fungal rRNA operon (White et al., 1990 (link)). The PCR and tag-encoded high-throughput sequencing of the ITS2 were performed using the Illumina HiSeq platform (PE 250) (Guangdong Magigene Biotechnology Co., Ltd., Guangzhou, China).

Total cellular RNA was extracted using RNAiso plus reagent (9,108, Takara, Kyoto, Japan) according to the manufacturer’s instructions. Subsequently, the total RNA concentration was determined with a NanoDrop 2.0 spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, United States) and the RNA was reverse transcribed to cDNA using a PrimeScript™ RT reagent kit with gDNA Eraser (RR047A, Takara) according to the manufacturer’s instructions. Subsequently, qRT-PCR assays were performed by using a SYBR Premix Ex Taq™ II (2×) kit (RR820A, Takara) according to the manufacturer’s instructions and run on an ABI 7500 Real-Time PCR Detection System (Foster City, CA, United States). The reactions were performed using the following parameters: 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The primer nucleotide sequences used for qRT-PCR are listed in Supplementary Table S1. All primer sets for mRNA amplification were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The relative expression levels of the target gene were normalized with respect to the levels of β-actin expression and calculated using the 2^−△△CT method.

Gene expression was analyzed with real-time reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from RAW264.7 cells using a RNeasy Mini Kit (Qiagen, Hilden, DEU). Total RNA concentration was determined with a NanoDrop 2.0 spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA), and 2 μg total RNA was reversibly transcribed into cDNA for 5 min at 70°C using a M-MLV cDNA Synthesis Kit (Enzynomics, Dajeon, KOR). PCR was performed using the TOPreal™ qPCR 2X PreMIX (Enzynomics, Dajeon, KOR) and run on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems 7500 System, Foster City, CA, USA; using Sequence Detection System software version 2.0.1). The relative mRNA levels were normalized using GAPDH as a housekeeping gene.