Primescrip rt reagent kit with gdna eraser

Total RNA from carnation leaves was extracted using the Plant RNA Kit (Omega, Norcross, GA, USA) according to the instructions. Subsequently, 500 ng of total RNA was reverse-transcribed to first-strand cDNA by the PrimeScrip RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s protocol, and the cDNA was diluted 10-fold for quantitative real-time PCR (qRT-PCR). qRT-PCR was performed using 2 μL of cDNA in a 20 μL reaction volume with SYBR^® Premix Ex Taq™ II (TaKaRa, Dalian, China) on a StepOnePlus Real-Time PCR System (ABI, USA), using the following PCR program: 95 °C for 3 min, followed by 40 cycles at 95 °C for 30 s and at 60 °C for 1 min 30 s. Melting curves were obtained to verify the amplification specificity through a stepwise heating of the amplicon from 60 to 95 °C. Primer pairs were designed by Primer Premier 5.0 (Table S4). The GAPDH gene was used as an internal control gene. Three independent biological replicates were performed, and the relative expression levels of the DcaHsf genes were calculated with the 2^−∆∆Ct method [53 (link)].

Total RNA was extracted with the RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). RNA integrity was evaluated using agarose gel electrophoresis and a NanoDrop spectrophotometer (Thermo Scientific, Los Angeles, CA, USA). A total of 1 μg high-quality RNA was used as the input material for cDNA synthesis with the PrimeScrip RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Inc., Dalian, China). Real-time quantitative PCR (qRT-PCR) was implemented on the ABI ViiA 7 DX system (Applied Biosystems) using TB Green Premix Ex Taq II (TaKaRa) with the ubiquitin (UBQ) gene as a reference gene to normalize expression data. The specific primer used for qRT-PCR was designed using Primer Premier 5 software and the sequence is listed in Table S1. Each PCR reaction contained 5.0 µL TB Green mix, 0.8 µL primers, and 1.0 µL diluted cDNA in a final volume of 10 µL. The amplification conditions were as follows: 30 s of denaturation at 95 °C, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s, then 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s. Each experiment was repeated at least triplicate and each gene was calculated with the 2^−ΔΔCT method for the relative expression.

Total RNA was extracted from cells using Trizol reagent (Sangon Biotech, China) according to the manufacturer’s instructions, followed by reverse transcription to cDNA using PrimeScrip™ RT reagent Kit with gDNA Eraser (Takara, Japan). All primers were synthesized by and purchased from the Tsingke Biotechnology Co., Ltd., China. Quantitative real-time PCR was performed using cDNA primers specific for mRNA (7500 Fast Real-Time PCR System, Applied Biosystems, USA). The real-time PCR reactions were performed using Takara’s SYBR Premix Ex Taq™ II (Tli RNaseH Plus) in the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The gene ACTB gene was used as internal control. The details of primer sequences are provided in Supplementary Table 1. Relative transcript abundance of a gene is expressed in ∆Ct values (∆Ct = Ct_reference – Ct_target). Relative changes in transcript levels compared with controls are expressed as ∆∆Ct values (∆∆Ct = ∆Ct_treated – ∆Ct_control). Experiments were performed in triplicate.

Biopsies were recovered from thawed OCT and RNA was extracted with the RNA/DNA/Protein Purification Plus Kit (Norgen Biotek). Tissue homogenization was made effective by the use of pestles. NanoDrop instrument (Thermo Scientific) was used for RNA quantification, then 62.5 ng of RNA were reverse transcribed with the PrimeScrip RT Reagent Kit with gDNA Eraser (Takara) according to the manufacturer's instructions. cDNA was diluted 1:2 with DNase and RNase-free water and stored at − 20° C.

Total RNA was isolated from cells using Trizol reagent (Takara, Japan). Reverse transcription was carried out with the PrimeScrip RT reagent Kit with gDNA Eraser (Takara, Japan). TB Green Premix Ex Taq kit (Takara, Japan) was used to perform real-time PCR, and we used the CFX96 Real-Time PCR Detection System (Bio-Rad, United States) with the following primers: HMSC GAPDH: forward, 5′-GGT​GGT​CTC​CTC​TGA​CTT​CAA​CA-3′ and reverse, 5′-TTG​CTG​TAG​CCA​AAT​TCG​TTG​T-3′; HMSC malat-1:forward, 5′- TCA​GGA​TAA​TCA​GAC​CAC​CAC​AG-3′ and reverse, 5′- GTA​ACT​ACC​AGC​CAT​TTC​TCC​AA-3′; Internal control was GAPDH.

For both mutants and their parents, ten mycelial plugs from 4-day-old cultures were added to 60 ml of potato dextrose broth in a 100 ml flask. The flasks were incubated for 24 h on a rotary shaker at 120 rpm. Either prochloraz (0.5 μg/ml), epoxiconazole (30 μg/ml), or difenoconazole (25 μg/ml) were added to the corresponding flasks; other flasks were free of fungicides. After 24 h, mycelia were harvested by vacuum filtration for RNA extraction (Fan et al., 2014 (link)). Total RNA was extracted using the SV Total RNA Isolation kit (Promega, Beijing, China), and cDNA was synthesized using the PrimeScrip RT reagent Kit with gDNA Eraser (Takara, Beijing, China) following the manufacturer’s protocol. Three independent experiments were conducted.
Real-time (RT)-PCR was performed with an ABI7500 sequence detection system (Applied Biosystems, United States). Amplifications were conducted using the SYBR Premix Dimer Eraser kit (Takara, Beijing, China). The relative quantities (RQ) of products were calculated using the 2^-ΔΔCt method. The β-tublin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used as references to normalize the quantification of CtCYP51 expression (Fan et al., 2014 (link)). The thermal cycling conditions and primers are listed in Supplementary Table S3.

Messenger RNA (mRNA) gene expression was assessed via RT-qPCR using 2 × TB Green Premix (TaKaRa, Japan) per manufacturer’s protocol. We prepared total RNA from E:H:p or A:H:p strains using a TRIzol Reagent Kit (Invitrogen Corp., Carlsbad, CA, USA). RNA concentration was measured on the Nanophotometer N60, and its integrity was validated by agarose gel electrophoresis, followed by complementary DNA (cDNA) synthesis using a PrimeScrip RT Reagent Kit with gDNA Eraser (TaKaRa). For qPCR, we used the chromosomal marker L-idonate and D-gluconate transporter (idnT) as the reference gene. The conditions for RT-qPCR are detailed in Text S3 in the Supplemental Methods. The relative expression level was determined using the comparative ^C_T (^ΔΔC_T) method [27 (link)]. The primers used are listed in Table S2.