Transscript one step gdna removal and cdna synthesis supermix

BIOER’s LineGene 9620 real-time fluorescence quantitative PCR system was used to select 10 genes for qRT-PCR analysis, and β-action was used as the reference gene (Table S11). These genes included StUSP2, StUSP6, StUSP13, StUSP14, StUSP15, StUSP21, StUSP24, StUSP9, StUSP33, and StUSP41; the primer sequences were designed using prime-blast [89 (link)]. Total RNA was extracted from each treated sample using the Total RNA Rapid Extraction Kit (ER501-01, TransGen Biotech, Beijing, China) and reverse-transcribed to cDNA using TransScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311, TransGen Biotech, Beijing, China). qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) kit. Three biological replicates were calculated using the 2^−∆∆Ct method [90 (link)].

Total RNA extraction and qRT-PCR analysis were performed as described by Guan et al. (55 (link)). Total RNA of the mycelia of the HN08 strain and mutants were extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). cDNA synthesis was performed with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The expression levels of the target genes were quantified by qRT-PCR performed with an ABI7500 sequence detection system (Applied Biosystems, Waltham, MA, USA). Reactions were performed in a total volume of 10 µL using the SYBR Premix Dimer Eraser Kit (Takara, Beijing, China). All of the reactions were repeated in at least three independent pools in three sets of biological replicates. The primer sequences were listed in Table S1.

The expression of keratin, vimentin, integrin β3, IL-2, TNFα, IFN-γ, IL-4, IL-10, VEGF, MMP9, and Ki-67 was assessed. Total RNA from rat uteri was isolated using TRI Reagent (Sigma) according to the manufacturer's protocol. Complementary DNAs (cDNAs) were synthesized using Trans Script One-Step gDNA Removal and cDNA Synthesis Super Mix (TransGen Biotech, Beijing, China). Real-time quantitative PCR was performed using primers and the Universal KAPA SYBR FAST qPCR Kit (Roche, Switzerland) in a 7900 Real-Time PCR System (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. All experiments were repeated at least three times. Primer sequences used in real-time quantitative PCR were showed in Table S2.

BmN cells were harvested at the corresponding time points and the total RNA was extracted using the TRIzol™ reagent ((TaKaRa, Dalian, China). Thereafter, the first-strand cDNAs was synthesized using 5 μg total RNA with the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). The primers are listed in Supplementary Table S2. qRT-PCR was performed using Hieff^® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). 95 °C for 5 min, then 40 cycles at 95 °C for 5 s, and 60 °C for 30 s were included in the reaction programs. Each sample was triplicated. The 2^−ΔΔCt method was applied to calculate the relative expression levels of selected genes.

Total RNA from the samples was extracted using the TRIzol (Trizol reagent 10296028, United States) method and the reverse transcription kit (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, AT311-03, TransGen Biotech, China) was used to synthesize first-strand cDNA and remove genomic DNA. Total RNA was stored in a −80 ± 1°C refrigerator prior to subsequent experimentation.

Total RNA was extracted from different parts (root, stem, leaf, flower, fruit, pollen grain and pollen tube) of pear using an RNA extracting kit (RNAsimply Total RNA Kit, Tiangen, Beijing, China) according to the manufacturer’s protocol. Then approximately 2 μg total RNA was used for first-strand cDNA synthesis using TransScript®One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANSGEN, Beijing, China). Three biological and three technical replicates were processed for the qRT-PCR assays, which was performed in 20 μL reaction mixture containing 80–100 ng cDNA, 200 nM of each primer (Additional file 1: Table S1) and 10 μL LightCycler 480 SYBRGREEN I Master Mix (Roche, Basel, Switzerland). All reactions were carried out in a CFX96 Real-Time System (Roche), following a three step standard protocol (45 cycles of 10 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C), followed by a melt curve analysis. The expression levels of PbrSLAC/SLAH genes were calibrated by the 2^–ΔΔCt method using PbrUBQ and PbrTUB as reference genes [35 (link)]. RT-PCR was carried out on a Veriti™ 96-Well Thermal cycler (Bio-Rad, Richmond, CA, USA) using Taq DNA Polymerase (New England Biolabs, Beverly, MA, USA). PbrUBQ was used as the reference gene. PCR products were separated on a 2% (w/v) agarose gel stained with ethidium bromide.

RT-qPCR was performed as previously described (Xu et al., 2019 (link)). Primers used for this assay were listed in Table S1. After the overnight culture was 1:50 diluted and grown to OD₆₀₀ of 1.0, 1 ml cell culture was collected. Total RNA was extracted using the Eastep^® Super Total RNA Extraction Kit (Promega) following the manufacturer’s instructions. Reverse transcription was conducted to obtain cDNA using the TransScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen). qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme). Relative gene expression was calculated based on the 2^-ΔΔCt method with the recA gene as the internal reference control (Schmittgen and Livak, 2008 (link)).