Hiscript 2 q rt supermix for qpcr gdna wiper reverse transcription kit

Quantitative real-time PCR was based on the method of Muhammad Ahsan Altaf [77 (link)], and some modifications were performed. According to the manufacturer’s instructions, total RNA was extracted from different treated tomato roots using an RNA simple total RNA extraction kit (TIANGEN, Beijing, China). With the help of agarose gel electrophoresis and a K5800 micro spectrophotometer (KAIAO, Beijing, China), the purity of the extracted RNA was detected. The RNA was then reverse transcribed using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme, Nanjing, China) for complementary DNA (cDNA) synthesis. For the qRT-PCR analysis, cDNA was used as a template, and the SYBR^®GREEN Premix Pro Taq HS qPCR Kit (ROX Plus) (Accurate Biotechnology, Changsha, China) was used in the QuantStudio5 P-qPCR system (Applied Biosystems, Waltham, MA, USA), using 96-well plates for the qRT-PCR.
The detailed information on the primers used in this study is provided in Table 2, and actin is used as the reference gene. The relative expression level of the gene was calculated using the 2^−ΔΔCt method by referring to the Livak [78 (link)] and Schmittgen [79 (link)] method.

The tissue samples from the wounded sites of Navel oranges were collected at 24, 48, and 72 hpi using 0 hpi as a control. The mycelium samples from PDA medium at 26 °C were collected at 24, 48, 72, and 96 hpi, respectively. P. italicum spores that were frozen immediately after culturing on PDA medium at 26 °C for one week were prepared as a control.
The total RNA of P. italicum mycelia and Navel orange tissues were extracted using an RNA kit (Omega Bio-tek, Doraville, GA, USA) and plant total RNA extraction kit, respectively, according to the manufacturer’s instructions. cDNA was synthesized with a HiScript ^®II Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme Biotechnology Co., Ltd., Nanjing, China), following the manufacturer’s protocols. Each experiment was repeated three times.

The muscle tissue of rare minnow from all treatment groups was homogenized in TRIZOL (Simgen, Hangzhou, China). Total RNAs were extracted with TRIZOL reagent, and converted to cDNA using the Reverse Transcription Kit HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co.,Ltd, Nanjing, China). All the cDNA concentrations were adjusted to 50 ng/μL. The qRT-PCR mixture consisted of 4 μL of cDNA sample, 3.1 μL of nuclease-free water, 7.5 μL of 2× SYBR Green master mix (No. BSB25L1B, BioEasy, Hangzhou, China), and 0.2 μL of each gene-specific primer (10 mM). The PCR cycling conditions were as follows: 1 cycle of 95 °C for 30 s, 45 cycles of 95 °C for 5 s, 60 °C for 30 s, 1 cycle of 95 °C for 15 s, 60 °C for 30 s, followed by dissociation curve analysis to verify the amplification of a single product. mRNA expression levels were normalized to the expression level of β-actin, and the data were analyzed using the 2^-△△CT method. The qRT-PCR primers are shown in Table 2.