ion period, the mycelium was scraped from the surface and collected below sterile conditions, speedily frozen in liquid nitrogen and stored at -80 C till RNA extraction. 4.six.2. RNA Extraction Frozen mycelium was used for RNA extraction using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich). RNA concentration ( /mL) and MMP-2 Purity & Documentation purity (A260/A280 ratio) were determined employing a 1.5- aliquot on a NanoDropTM spectrophotometer (Thermo Fisher Scientific, Madrid, Spain). Samples were diluted to 0.1 / and treated with DNAse I (Thermo Fisher Scientific) to eliminate genomic DNA traces that could possibly be co-extracted with RNA. 4.six.3. Two-Step Reverse-Transcription Real-Time PCR Retrotranscription Reaction Synthesis of complementary DNA (cDNA) was carried out utilizing 5 of total RNA in accordance with the manufacturer’s guidelines of the PrimeScriptTM RT reagent Kit (Takara Bio Inc., Kusatsu, Shiga, Japan). The reaction conditions had been incubation at 37 C for 15 min and reverse transcriptase inactivation at 85 C for five s. Then, cDNA samples were stored at -20 C until gene expression evaluation. Real-Time PCR Reactions The real-time PCR (qPCR) reactions have been carried out in a 7300 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) utilizing SYBRGreen technology. The amplification of aflR and -tubulin genes was conducted according to the methodology described by Peromingo et al. [48]. Briefly, the final volume of the reaction mixture for the amplification of every gene was 12.five and consisted of 6.25 of SYBRPremix Ex TaqTM (Takara Bio Inc., Kusatsu, Japan), 0.05 of ROX plus (Takara Bio Inc.) and 2.5 of cDNA template. For the aflR gene, the final concentration from the primer pair AflRTaq1/AflRTaq2 was 300 nM every, while that on the primers F-TUBjd/R-TUBjd used to amplify the -tubulin gene was 400 nM each and every. The thermal cycling situations for amplification of both genes included a single initial denaturation step at 95 C for 10 min, and 40 cycles at 95 C for 15 s and 60 C for 30 s. After the final PCR cycle, melting curve analyses in the PCR merchandise were conducted and checked to ensure the fidelity of the final results. The quantification cycle (Cq), the cycle in which fluorescence reaches a defined threshold, was automatically calculated by the instrument working with the default parameters of your 7300 Rapidly Technique Computer software (Applied Biosystems). four.6.four. Calculation of Relative Gene Expression Relative quantification from the expression from the aflR gene was basically STAT6 manufacturer performed as previously detailed by Peromingo et al. [48]. The expression ratio was calculated making use of the 2-CT approach [56]. The -tubulin gene was made use of as an endogenous manage. Calibrators corresponded for the A. flavus strain grown inside the absence of yeast (batch AF, handle), and the samples had been incubated for 3 days (1st sampling day). four.7. Aflatoxin Analysis Aflatoxin extraction was performed per the method described by Ruiz-Moyano et al. [57], with some modifications. The content material of one particular Petri dish was transferred to a filter plastic bag and macerated with one hundred mL of chloroform within a Stomacher Lab-Blender 400 (Seward Medical, Worthing, UK) for 2 min. After 1 h in darkness at space temperature, the slurry was filtered twice by means of anhydrous sodium sulphate with Whatman no. 1 filter paper (Whatman International, Maidstone, UK). Then, the filtrate was evaporatedToxins 2021, 13,14 ofin a rotatory evaporator model Hei-Vap (Heidolph, Schwabach, Germany) at 37 C. The residue was resuspended in six mL of chloroform, transferred