Eastep total rna extraction kit

Total RNA was extracted from the cells/liver tissues using Eastep™ Total RNA Extraction Kit (Promega, Madison, United States) and miRNAs were extracted from the cells/liver tissues using the EasyPure miRNA Kit (TransGen, Beijing, China) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen) to detect mRNA. cDNA was generated using a Ribo SCRIPT™ Reverse Transcription kit (RiboBio) to detect miRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using PerfectStart™ Green qPCR SuperMix (TransGen). β-actin was used as an mRNA control. U6 was used as a reference miRNA control. The primers for U6/miR-654-5p were obtained from RiboBio Co., Ltd. All other qPCR primers used are listed in Table 1 qPCR was performed using the Agilent Mx3005P Real-Time PCR System (Applied Biosystems, Foster City, CA).

Expression levels of genes involved in asexual development and AF biosynthesis were measured by RT-qPCR. The 48 h-old liquid shaken mycelium were harvested from PDA medium and lyophilized for the preparation of total RNA extraction. RNA was extracted from 100 mg of indicated mycelium using the Eastep Total RNA Extraction Kit (Promega, Madison, WI, USA) and treated with RNase-free DNase I (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). To do RT-qPCR reaction, SYBR Green Supermix (Takara) was used and detected with the PikoReal 96 Real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using the program of an initial denaturing step at 95 °C for 5 min followed by 40 cycles, each consisting of denaturing at 95 °C for 5 s and extension at 60 °C for 30 s. The primers used for RT-qPCR are listed in Table 4. The efficiency of all the primers was between 90% and 110%.

Total RNA was extracted using the Promega Eastep™ Total RNA Extraction Kit (Promega, Beijing, China) according to the manufacturer’s instructions. The RNA concentrations were detected using NanoPhotometer™ Microvolume Spectrophotometer (IMPLEN, Beijing, China). First-strand cDNA synthesis was performed using the UnionScript First-strand cDNA Synthesis Mix for qPCR Kit (Jinsha, Beijing, China) following the manufacturer’s instructions. Quantitative RT-PCR analysis was performed according to the manufacturer’s instructions for an aq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China) using an ABI 7300 Real-Time system (Bio-Rad, Hercules, CA, USA).

RNA was extracted with Eastep™ total RNA extraction kit (Promega, United States) and reverse transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, United States) following the manufacturer’s instructions. PCR was performed with 2×EasyTaq^® PCR SuperMix (TransGen Biotech, China). Amplification was performed as follows: a denaturation step at 94°C for 5 min, followed by 30 cycles of amplification at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s. Three parallel reactions were performed on GeneAmp^® PCR System 9,700 (Applied Biosystems, United States). GAPDH was used as a reference gene. Primers used in the RT-PCR were as follows: UBE2C FP: 5′- GGA​TTT​CTG​CCT​TCC​CTG​AA-3′, UBE2C RP: 5′-GAT​AGC​AGG​GCG​TGA​GGA​AC-3′, MALAT1 FP: 5′-GCG​ACG​AGT​TGT​GCT​GCT​ATC​T-3′, MALAT1 RP: 5′-ACA​CTG​CTC​TGG​GTC​TGC​TTT​T-3′, GAPDH FP: 5′-CTC​CTC​CTG​TTC​GAC​AGT​CAG​C-3′, GAPDH RP: 5′-CCC​AAT​ACG​ACC​AAA​TCC​GTT-3′.

The WT, ΔaflE, ΔaflE::aflE, K370A, and K370R strains were further verified by Southern blot with the North2South™ Biotin Random Prime DNA Labeling Kit (No. 17075, Thermo Scientific) and North2South™ Chemiluminescent Hybridization and Detection Kit (No. 17097, Thermo Scientific), according to a previous study (56 ). Simply, genomic DNA from each strain was singly digested with EcoR I and hybridized with a 0.959 kb probe of the upstream region fragment of aflE. For quantitative real-time PCR, mycelia were harvested after incubation in YES media at 28 °C for 48 h in the dark, and immediately ground in liquid nitrogen. RNA was isolated from 100 mg of ground mycelia with the Eastep Total RNA Extraction Kit (Promega) and purified with RNase-free DNase I (Thermo Scientific). cDNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Subsequently, qPCR was performed on a PikoReal Real-Time PCR machine (Thermo Scientific) using the SYBR Green qPCR mix (TAKARA) (primers listed in supplemental Table S1). The expression of the aflE gene was analyzed and the actin gene was used as an endogenous control. The REST 2009 software was used to calculate the relative expression of target genes with the PairWise Fixed Reallocation Randomization Test (57 (link)).

Total RNA was isolated from the cultured cells using an Eastep Total RNA Extraction kit (Promega) according to the manufacturer’s protocol. Then, total RNA was reverse transcribed using a TransScript First-Stand cDNA Synthesis kit (TransGen Biotech). RT-qPCR was subsequently performed with TransStart Green qPCR SuperMix (TransGen Biotech), and products were detected with a DA7600 Real-time Nucleic Acid Amplification Fluorescence Detection System (Bio-Rad, Hercules, CA, USA). We quantified the transcripts of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal mRNA quantity control. All primers for cDNA amplification of various target genes were designed and optimized using Oligo 7.0 software (Molecular Biology Insights, West Cascade, USA) and synthesised by Sangon Biotech (Shanghai, China). The relative expression levels of the target genes were calculated by normalizing the cycle threshold (Ct) values of the target gene to the Ct values of GAPDH (ΔCt) and determined as 2^-ΔCt. The RT-qPCR products were subjected to electrophoresis.

Total RNA was extracted using the Eastep Total RNA Extraction Kit (Promega, Beijing, China), and cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan). cDNA was diluted to 100 ng/µL. qRT-PCR was performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Shiga, Japan) on the CFX384 Touch Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each 20 µL qRT-PCR reaction contained 10 µL 2x SYBR Premix Ex Taq mix, 0.2 mM of each primer, and 2 µL cDNA. Primers for each gene are shown in Table S1. MtActin and AtActin were used as the internal reference genes for M. truncatula and Arabidopsis samples, respectively. Relative gene expression was calculated using the 2^−ΔΔCt method [65 ]. There were three technical replicates for each experiment.