Lightcycler

mRNA isolated via TRAP was DNAse treated (Promega, M6101) for 30 min at 37°C. cDNA was synthesized using SuperScript III First-Strand Synthesis kit (ThermoFisher, 18080051) according to manufacturer specifications. Sybr green (Roche) qPCR master mix was used to quantify il1b, cxcl8a, cxcl8b.1, and tnfa gene expression in addition to rps11 expression as a normalization control (Table 1). qPCR reactions were run on a Lightcycler (Roche) and Cq values were quantified from Lightcycler software and normalized to housekeeping gene rps11.

Plasma folate and homocysteine levels were measured using commercially available kits by fluorescence polarization immunoassay (Becton Dickinson, Franklin Lakes, NJ, USA) on an Abbott 130 AxSYM system (Becton Dickinson). Tissue folate was quantified using a microbiological assay by glycerol-protected Lactobacillus casei (BCRC^®10697) in 96-well microtiter plates as previously described elsewhere [29 (link)]. Lymphocytic DNA was extracted using standard proteinase K digestion and the phenol–chloroform extraction procedure. The MTHFR C677T polymorphism was determined through RT-PCR and melting curve analysis by using a LightCycler instrument (Light-Cycler, Roche Diagnostics, Mannheim, Germany) as previously described [30 (link)]. Bisulfite modification of lymphocytic DNA was performed using the EpiTect PCR Control DNA set (Qiagen, Hilden, Germany), and the high-resolution melt-based PCR method was used to measure DNA methylation. Primers used for LINE-1 were F 5′-GCG AGG TAT TGT TTT ATT TGG GA-3′ and R 5′-CGC CGT TTC TTA AAC C-3′ to encompass eight CpG islands between primers and yield 141 bp of amplicon size. RT-PCR was conducted by use of a LightCycler instrument (Light-Cycler, Roche Diagnostics, Mannheim, Germany).

Total RNA was extracted from cells and exosomes by the mirVana™ miRNA Isolation Kit (Ambion Inc.). Retrotranscription and amplification were performed using Transcription first-strand cDNA Synthesis Kit and LightCycler 480 SYBR Green I Master Kit in a Light-Cycler apparatus (Roche Diagnostics). For PCR Arrays analysis we used RT² PreAMP CDNA Synthesis Kit and RT² PreAMP Pathway Primer Mix for Stem Cell Transcription Factors and for Human Tumor Metastasis, in the appropriate PCR Arrays following the manufacturer's instructions (Qiagen). In the validation set, retrotranscription was achieved as described above. Next, we performed a specific pre-amplification using Real-Time Ready cDNA PreAMP Master using PreAMP Primer Pools, followed by Real-Time Ready Custom Panels in a Light-Cycler apparatus (Roche Diagnostics) in line with the manufacturer's instructions.
cDNAs were quantified by NanoDrop ND-1000 (Thermo Scientific); and concentrations were equalized to 100 μg/μL to standardize per cDNA amount.
The sequences of the primer sets and the reaction conditions are shown in Supplementary Table S5.

Total RNA was isolated using the RNeasy Mini Kit (EZ™ Total RNA Mini Prep Kit, Enzynomics, South Korea) according to the manufacturer’s protocol and reverse transcribed using the HB_I RT Reaction Kit. cDNAs were amplified by qRT-PCR using the synthesized specific HB_I Nucleic Mix II primers and RNU6B HB primers (HeimBiotek, South Korea). PCR was performed using the LightCycler instrument (Roche Applied Sciences, Indianapolis, IN, USA). PCR was started at 95°C for 15 min, followed by 40 cycles at 95°C for 10 s and 60°C for 40 s, and finished with 95°C for 60 s, 55°C for 30 s and 95°C for 30 s. The expression of RNU6B was used to normalize the expression of target genes. The specific primer Has-miR-216b was designed and synthesized by HeimBiotek Company (HeimBiotek, South Korea). Relative miRNA fold change was normalized using standard C_t values of RNU6B (U6) (HeimBiotek, South Korea). RT-PCR was performed using the LightCycler instrument (Roche Applied Science, Indianapolis, IN, USA).

Effect of AECS on the expression of hyphal specific genes, hyphal wall protein 1 (HWP1), Agglutinin-like protein 1 (ALS1) and fourth secreted aspartyl proteinase (SAP4), was evaluated by qRT-PCR. Hot phenol/chloroform extraction method⁵¹ was used in extraction of total RNA from AECS treated (80 μg/ml) and untreated (0 μg/ml) hyphal growth of selected strain. Quantitative RT-PCR amplification mixtures (25 ml) contained 10 ng template cDNA, Light Cycler Hybridization Probes Master Mix kit (Roche diagnostics, Tenay, Turkey), and SYBR Green I master mix buffer with fluorescein. Light Cycler (Roche diagnostics, Tenay, Turkey) and Light Cycler 3.5 software were used^{52 (link),53} .