Total RNA was isolated from cells and EVs using Trizol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). Concentration and quality of RNA was measured by Nanodrop2000 (Thermo Fisher). miRNAs were reverse transcribed using the HifairTM II 1st Strand cDNA Synthesis Kit (Yeasen). Quantitative real-time PCR analysis was performed in ABI7500 real-time PCR amplifier (Applied Biosystems, Waltham, MA, USA) using SYBR Green Master Mix (Yeasen). U6 or cel-miR-39 was used as control, and results were analyzed using the 2−ΔΔct calculation method.
Abi7500 real time pcr amplifier
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
The ABI7500 real-time PCR amplifier is a laboratory instrument designed for the amplification and detection of nucleic acid sequences using the real-time PCR (Polymerase Chain Reaction) technique. The core function of the ABI7500 is to precisely control the temperature and cycling of the PCR reaction, as well as to detect and measure the fluorescent signals generated during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Hifairtm 2 1st strand cdna synthesis kit, by Yeasen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Yeasen (2 mentions)
E z n a plant rna kit, by Omega Bio-Tek (2 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Kapa sybr fast qpcr kit master mix, by Roche (1 mentions)
E z n a tm plant rna kit, by Omega Bio-Tek (1 mentions)
Sybr premix ex taqtm 2 kit, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Dna extraction kit, by Promega (1 mentions)
Braf mutant gene detection kit, by Amoy Diagnostics (1 mentions)
Mirneasy serum plasma spike in control, by Qiagen (1 mentions)
Miscript sybr green, by Qiagen (1 mentions)
Miscript primer assay, by Qiagen (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Chamq sybr qpcr master mix kit, by Vazyme (1 mentions)
10 protocols using abi7500 real time pcr amplifier
1
RNA Extraction and miRNA Quantification
2
RNA Extraction and qPCR Analysis for Mycelia
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Total RNA was extracted from the mycelia using E.Z.N.A.TM Plant RNA Kit (Omega Bio-Tek) according to the manufacturer’s instructions. Briefly, 150 ng total cellular RNA was reverse transcribed using TIANScript RT Kit. The KAPA SYBR FAST qPCR Master Mix Kit (Kapa Biosystems, United States) and the ABI 7500 Real-Time PCR amplifier (Applied Biosystems, Foster City, CA, United States) were used for qPCR. All reactions were carried out in a total volume of 20 μL which contained 2 μL of diluted cDNA, 0.8 μL of primer mix (10 μM), 6.8 μL of nuclease-free water, 0.4 μL ROX Low, and 10 μL of SYBR Green mix. All reactions were performed in triplicate. The qPCR amplification procedures were as follows: 95°C for 3 min, 40 cycles of 95°C for 3 s, 60°C for 32 s, and a final extension at 72°C for 30 s. The GAPDH-encoding gene, gapdh, was used as the reference. Primers were designed using the DNAMAN software (Table 2 ) and were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
3
Quantification of TMEM40 mRNA Expression
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To quantify TMEM40 mRNA expression, total RNAs were isolated from the cells 48 h after transfection. Total RNAs were extracted using RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol and dissolved into nuclease-free water. A PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) was used for RT of mRNA to cDNA. Then, qPCR was performed to detect TMEM40 expression levels using the SYBR® Premix Ex TaqTM II kit (Takara Biotechnology Co., Ltd.) and an ABI 7500 real-time PCR amplifier (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermal cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 34 sec. The following primers were used: TMEM40 forward, 5′-CAGAGCAACCGGAAAACATCG-3′ and reverse, 5′-CTGGGCTACACTGAGCACC-3′; GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAATG-3′. Each reaction was analyzed in triplicate. The comparative Ct (2−ΔΔCq) (10 (link)) method was used to calculate the relative levels of TMEM40.
4
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from cells and EVs using Trizol reagent (Invitrogen, Life Technologies, USA). The concentration and quality were assessed using the Nanodrop2000 (Thermo Fisher Scientific). RNA was reverse transcribed using the HifairTM II 1st Strand cDNA Synthesis Kit (Yeasen, China). Quantitative real-time PCR analysis was performed on the ABI7500 real-time PCR amplifier (Applied Biosystems, USA) using SYBR Green Master Mix (Yeasen, China). U6 or cel-miR-39 was used as a control, and results were analyzed using the 2–ΔΔct method.
5
Detecting BRAF V600E Mutation in PTC
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Determination of the BRAFV600E gene mutation was carried out for all patients with PTC by PCR techniques (20 (link)). DNA was isolated from PTC tissues using a DNA Extraction kit (Promega Corporation, Madison, WI, USA), and BRAF gene exon 15 was detected using a BRAF mutant gene detection kit (Amoy Diagnostics Co., LTD, Fujian, China) according to the manufacturer's protocol and the ABI7500 real-time PCR amplifier (Applied Biosystems; Thermo Fisher Inc., Waltham, MA, USA). The primers for amplification of exon 15 of BRAF were designed as follows: Forward, 5′-TCATAATGCTTGCTCTGATAGGA-3′ and reverse, 5′-GGCCAAAAATTTAATCAGTGGA-3′). All procedures and analyses were carried out in the biomolecular laboratory of the Hongqi Hospital Affiliated to Mudanjiang Medical University.
6
Quantitative Analysis of miRNA and hY4 RNA
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Spike-in control cel-miR-39 mimic (miRNeasy Serum/Plasma Spike-In Control, Qiagen) was added to total RNA samples as internal control, and the mixed RNA was reverse-transcribed to cDNA using a reverse transcriptase kit (miScript II RT Kit, Qiagen). SYBR-based qRT-PCR was performed on an ABI7500 real-time PCR amplifier (Applied Biosystems) using specific primers (miScript primer Assay, Qiagen) and a universal PCR kit (miScript SYBR Green, Qiagen) according to the manufacturer’s protocol. For hY4 RNA fragments detection, U6 snRNA was used as internal control. Amplifying conditions were as follows: 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 34 s [84 (link), 85 (link)]. Gene expression data were normalized to internal control expression, and the relative expression was determined as 2−Δ Ct, where Δ Ct = Ct (target gene) − Ct (internal control).
7
Quantifying Fungal Gene Expression via qPCR
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The levels of specific mRNAs expressed by the WT and transformed strains were assessed using qPCR according to methods in a previous study (3 (link)). A fusion fragment containing the gpd promoter and aco gene was amplified using the primer pair gpd+aco-F and gpd+aco-R (Table 1 ). Based on a previous description, total RNA was extracted using the E.Z.N.A. plant RNA kit (Omega Bio-tek, Norcross, GA, USA) following an extraction method for fungal samples. First-strand cDNA was synthesized using the HiScript II 1st strand cDNA synthesis kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The ChamQ SYBR qPCR master mix kit (Vazyme, Nanjing, China) and the ABI 7500 real-time PCR amplifier (Applied Biosystems, Foster City, CA, USA) were used for qPCR. The qPCR amplification procedure was as follows: 95°C for 3 min, 40 cycles at 95°C for 3 s and at 60°C for 32 s, and a final extension at 72°C for 30 s. Relative gene expression was analyzed according to the 2−△△CT method (55 (link)).
8
Quantifying TMEM40 mRNA Expression
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To determine TMEM40 mRNA expression levels, total RNA was extracted from clinical samples or from A431 and SCL-1 cells 48 h after transfection using a PureLink RNA Mini kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was quantified using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). A TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to reverse transcribe cDNA from total RNA according to the manufacturer's instructions. SYBR-Green PCR kit (Takara Biotechnology Co., Ltd.) and an ABI 7500 realtime PCR amplifier (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used for qPCR. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 34 sec. GAPDH was used for normalization. The relative gene expression data were detected by qPCR and analysed with the 2−ΔΔCq method (19 (link)). The RT-qPCR primers and their sequences are listed in Table I .
9
Quantitative Analysis of Antioxidant Genes
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Total RNA was extracted using an E.Z.N.A. Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA) following an extraction method for fungal samples. First-strand cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The ChamQ SYBR RT-qPCR Master Mix Kit (Vazyme, Nanjing, China) and the ABI 7500 real-time PCR amplifier (Applied Biosystems, Foster City, CA, USA) were used for RT-qPCR. According to our previous study [81 (link)], the RT-qPCR amplification procedure was as follows: 95 °C for 3 min, 40 cycles at 95 °C for 3 s and at 60 °C for 32 s, and a final extension at 72 °C for 30 s. In this study, RT-qPCR was performed to analyze the mRNA expression levels of the aox gene and key antioxidant enzyme genes in the CCMSSC00389 strain and aox-transformed strains subjected to the different treatments. The β-actin and β-tubulin genes were used as internal reference genes, and the relative gene expression was determined according to the 2−△△CT method.
10
Quantifying miRNA Expression by Stem-Loop RT-qPCR
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The expression levels of the milRNAs were quantified by stem-loop real-time PCR, using 5S rRNA as the internal control for each sample (Zhou Q. et al., 2012 (link)). The stem-loop primers in the reverse transcription kit and the upstream primers used for qRT-PCR were designed using miRNA design software.3 The first-strand cDNA was synthesized using a miRNA first Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions. The miRNA Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and an ABI 7500 real-time PCR amplifier (Applied Biosystems, Foster City, CA, United States) were used for qRT-PCR, as described in our previous study. The expression of the target genes of the milRNAs was detected using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control for each sample, as previously described (Hou et al., 2021 (link)). The relative expression levels of the milRNAs and their target genes in the different stages were quantified using the comparative threshold cycle (CT) 2−△△CT method. The primers used for qRT-PCR amplification of the milRNAs are enlisted in Supplementary Table S1 .
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