Cfx connect thermal cycler

RNA was extracted from P. digitatum cells exposure to citral at various concentrations (0 and 1/2MIC) for 0, 30, 60, and 120 min using the Trizol reagent (Invitrogen, USA) following the manufacturer’s instructions. Two micrograms of DNA-free RNA were used for reverse transcription by M-MLV (Promega, USA) with oligo dT18. RTFQ-PCR was performed on a BIO-RAD CFX Connect Thermal Cycler using FastStart Universal SYBR Green Master (Roche, Switzerland). All primer pairs for expression assays are listed in Table 1. RTFQ-PCR was programmed as follows: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The 2^-△△CT method was used to quantify the value of every sample using actin gene as an internal reference [27 (link)].Table 1

Primer pair sequences designed for validation of differentially expressed genes in CK30 and T30 treatment P. digitatum using Real-time Fluorescence Quantitative PCR (RTFQ-PCR)

Gene ID	Gene name	Primer sequence (5′-3′, forward/reverse)
Unigene6144	Actin	TGCGCTGAACCGAACTGCCGTCGGGAGCCTCGAAGCGCTC
Unigene8313	ERG7	GCGCTGGCGATTGGTCGATG
		CAGGCCCAGTTTCCGGGCTCC
Unigene8539	ERG11	CCATCGACCTCGTCCCCGCC
		TCGCGCTTGCGGTTTTGGGG
Unigene6797	ERG6	CGCGTGATGCCGCCTTCAAC
		TGAGCCTTGCGGGCCTCACG
Unigene5828	ERG3	CAGGCCATGGCCGCAATGCC
		GGTGCAGGCCACGGTGGATCC
Unigene8125	ERG5	TCTCGCCATTGGCGGATGCG
		TCTCGCCATTGGCGGATGCG

RNA of P. digitatum treated with citronellal (0 or 1/2 MIC concentration) for 0, 30, and 60 min was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The expression levels of the ERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG7, ERG9, ERG11 ERG24, ERG25, ERG26, and ERG27 genes were examined using RTFQ-PCR. RTFQ-PCR was performed through a BIO-RAD CFX Connect Thermal Cycler using FastStart Universal SYBR Green Master (Roche, Switzerland) with the following programs: 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s, and at 60 °C for 1 min. Actin gene was used as internal reference, and the samples were quantified by 2^−△△CT method [31 (link)]. All primer pairs for expression assays are listed in Table 1. The experiments were repeated with addition of exogenous ergosterol (150 mg/L) to the fungal culture medium.