4.5. Gene Expression Analysis of OTA Cluster Genes in ∆otaY Mutant

An end-point Reverse Transcription (RT)-PCR and qRT-PCR were used to confirm the expression of OTA biosynthetic genes (otaA, otaB, otaC, otaR1 and otaD) and the absence of otaY transcript in deletion strain.The wild-type ITEM 5010 and the selected ∆otaY strain AC2021 of A. carbonarius were grown on MM agar plates for 4 days in the dark at 25 °C. Total RNA and cDNA were obtained as described above (Chapter 4.2). PCR amplifications were performed with Platinum SuperFi II PCR Master Mix (Invitrogen) on 100 ng of cDNA and 500 nM of each primer. PCR amplification conditions were: 98 °C for 30 s, then 30 cycles of 98 °C for 10 s, 58 °C for 15 s and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The primers Bt2a/Bt2b [43] were used to monitor β-tubulin gene expression as endogenous control. These primers span three introns, which also allowed checking for DNA contamination. Real-time PCR analysis was performed as reported in chapter 4.2. The relative quantification (2^−ΔΔCT) of gene expression was assessed by Quant-Studio™ RT-PCR Software (Thermo-Fisher Scientific, Waltham, MA, USA), considering as reference condition the expression level of each gene in wild-type strain ITEM 5010.