Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific). Complementary DNA was synthesised from 1 μg of total RNA using AccuPower RT PreMix Kits (Bioneer, Daejeon, Korea). Real-time PCR analysis was performed using TB Green™ Premix Ex Taq II Kit (TaKaRa, Tokyo, Japan) and AB7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Each sample was analyzed in quadruplicate, and target genes were normalized to the reference housekeeping gene Gapdh. Fold differences were then calculated for each group using CT values normalized to those of the control groups. The sequences of all primers used are listed in Table 1 .
Accupower rt premix kit
Manufactured by Bioneer
Sourced in United States, Germany
The AccuPower RT PreMix kit is a ready-to-use solution for reverse transcription (RT) reactions. It contains all the necessary components, including reverse transcriptase enzyme, buffer, and dNTPs, to efficiently convert RNA into cDNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Kod dna polymerase, by Merck Group (3 mentions)
Kod polymerase buffer, by Merck Group (3 mentions)
Vector nti advance 9, by Thermo Fisher Scientific (3 mentions)
Lightcycler faststart dna master sybr green kit, by Roche (3 mentions)
Lightcycler instrument, by Roche (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Tb greentm premix ex taq 2 kit, by Takara Bio (2 mentions)
Rnase mini kit, by Qiagen (2 mentions)
Oligo dt primer, by Thermo Fisher Scientific (2 mentions)
Accupower 2x greenstartm qpcr master mix, by Bioneer (2 mentions)
Trizol reagent, by Solgent (2 mentions)
Faststart essential dna green master kit, by Roche (2 mentions)
Accupower pcr premix kit, by Bioneer (2 mentions)
Lightcycler software, by Roche (2 mentions)
Trisure reagent, by Meridian Bioscience (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Ab7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq 2 kit, by Takara Bio (1 mentions)
38 protocols using accupower rt premix kit
1
Real-Time PCR Gene Expression Analysis
2
Quantitative Gene Expression Analysis
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The mice sacrificed by anesthesia using CO2. Total RNA from mouse livers was extracted using the TRIzol reagent. Concentrations of RNA and A260/A280 ratios were determined by spectrophotometry. The extracted RNA was reverse transcribed according to the manufacturer’s instructions. Complementary DNA was synthesized from 1 μg of total RNA using AccuPower RT premix kits (BIONEER, Korea). RT-PCR analysis was performed using the TB GreenTM Premix Ex Taq II kit (TAKARA, Japan) and iQ5 real-time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA) following the manufacturer’s instructions. Each sample was analyzed in triplicate, and the target genes were normalized to the reference housekeeping gene gapdh. The reaction conditions comprised pre-denaturation at 95 °C for 10 min, followed by 50 cycles of 5 s at 95 °C and 45 s at 55 °C. Table 1 showed the PCR primer sequences which were synthesized by Genechem Co., Ltd (China). The results were calculated using the conventional 2-△△Ct method.
3
Quantification of LC3B, TLR4, and TLR9 Transcripts
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Immediately after intracardiac perfusion, tissue samples from the left upper lobe were freeze-clamped in liquid nitrogen and stored at −80°C. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, USA). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using AccuPower RT premix kits (BIONEER, Korea). Real-time PCR analysis was performed using the TB GreenTM Premix Ex Taq II kit (TAKARA, Japan) and QuantStudio3 (Applied Biosystems, USA) according to the manufacturer’s instructions. Each sample was analyzed in quadruplicate, and target genes were normalized to the reference housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold differences were calculated for each group using the normalized CT values for the sham groups. The primer sequences for real-time PCR are as follows: LC3B: Forward 5’-AGAGCGATACAAGGGTGAGA-3’, Reverse 5’-ACTTCAGAGATGGGTGTGGA-3’; TLR 4: Forward 5’-CCAGAGCCGTTGGTGTAT CT-3’, Reverse 5’-TACAATTCGACCTGCTGCCT-3’; TLR 9: Forward 5’-AAATCGTTCAGTGAGC TCCC-3’, Reverse 5’-CTGAAGTTGTGGCCTATCCC-3’; GAPDH: Forward 5’-CTGAGAATGGGAAGCTGGTC-3’, Reverse 5’-CTCCACGACATACTCAGCAC-3’.
4
Cloning and Verification of hRANKL cDNA
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The RNA used to clone hRANKL cDNA was extracted from MG63 cells (ATCC ® CRL-1427 ™ ) expressing RANKL. The quality of the extracted RNA was veri ed by agarose gel electrophoresis. The cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea), according to the manufacturer's instructions. Ampli cation and cloning of the hRANKL fragment were carried out in a reaction mixture comprising KOD polymerase buffer, 10 mM dNTPs, 25 mM magnesium chloride (MgCl 2 ), 10 μM primers (hRANKL-BclI: 5'-TGATCAAAGCTTGAAGCTCAGCCTTTTGC-3' and hRANKL-XhoI: 5'-CTCGAGATCTATATCTCGAACTTTAAAAGCCCC-3'), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA) and 2 μL of the RANKL gene construct (template). The thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 70°C for 30 s. The polymerase chain reaction (PCR) product was cloned into the BamH1/XhoI sites of a pMX vector (CELL BIOLABS, USA). Sequence analyses were carried out using programs in Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA).
5
Genomic DNA Purification and Transgene Detection
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Genomic DNA from cells was purified using a QIAamp DNA Micro Kit (Qiagen, Valencia, CA, USA). The inserted transgenes were detected by PCR with genomic DNA (50 ng), using specific primers (F: 5′-GGA ACT CTT GTG CGT AAG TCG ATA G-3 and R: 5′-GGA GGC GGC CCA AAG GGA GGA GAT CCG-3). PCR was conducted using 35 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 40 s). After total RNA was prepared using an RNase Mini Kit (Qiagen), 1 μg of RNA was reverse-transcribed and used for qPCR using the AccuPower RT PreMix kit (Bioneer, Daejon, Korea) and FastStart Essential DNA Green Master (Roche, Indianapolis, IN, USA). GAPDH expression was used to normalize the mRNA expression levels. The primer sequences used are listed in Table 1 .
6
Yeast RNA Isolation and cDNA Synthesis
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A mini-prep version of yeast RNA isolation was performed with slight modifications [22 (link)]. Cells grown in YM medium were washed and resuspended in 400 μL of AE buffer (50 mM Na acetate pH 5.3, 10 mM EDTA). The resuspended cells were transferred to a microcentrifuge tube and 40 μL of 10% SDS, 440 μL of buffer-equilibrated phenol (Bioneer, Daejeon, Korea), and glass beads (Sigma) were added. The aqueous phase was extracted with 400 μL of phenol (pH 5.2):chloroform:isoamyl alcohol (25:24:1) (Bioneer) by centrifugation (13,000×g, 25 °C for 5 min). The aqueous phase was added to 40 μL of 0.3 M Na acetate, pH 5.3, after which 2.5 volumes of 95% ice-cold ethanol were added. The tube was incubated for 30 min at -20 °C and centrifuged at 4 °C for 5 min. The pellet was dried at 60 °C for 5 min and resuspended in 55 μL of sterile water and stored at − 70 °C until use.
For cDNA synthesis from total RNAs, 400 pmol of oligo dT was incubated with extracted RNAs at 70 °C for 5 min. The mixture was then chilled rapidly on ice. Each cDNA was synthesized (AccuPower RT PreMix kit, Bioneer) and used in subsequent PCR with specific primers to amplify the protein-coding regions.
For cDNA synthesis from total RNAs, 400 pmol of oligo dT was incubated with extracted RNAs at 70 °C for 5 min. The mixture was then chilled rapidly on ice. Each cDNA was synthesized (AccuPower RT PreMix kit, Bioneer) and used in subsequent PCR with specific primers to amplify the protein-coding regions.
7
Total RNA Extraction and cDNA Synthesis
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Total RNA of cells was extracted by DenaZist kit (Iran) under the manufacture protocol. Briefly, each sample was lysed and homogenized in 1 ml G1 buffer. The homogenate was incubated at room temperature (20°C) for 10 minutes. Then, 200 μl chloroform was added and sample was centrifuged at 12,000 g for 15 min at 4°C and upper phase containing RNA was precipitated with equivalent of half the volume aqueous phase of the isopropyl alcohol and the same volume from G2 buffer. Afterwards washing was performed by 75% ethanol and sample was dried in contact with air, and resuspended in diethyl pyrocarbonate (DEPC)-treated water. In order to remove any possible genomic DNA, five unit RNase free DNAse I (Roche, Germany) was added per each 20 μg of RNA and incubated at 34°C for 20 min followed by adding 0.8 μl 0.5 M EDTA and heat inactivation of the enzyme at 75 °C for 10 min. RNA concentration, purity and quality were appraised using NanoDrop 2000 (Thermo Scientific, USA) and gel electrophoresis. Then cDNA was synthesized by AccuPower® RT Premix kit (Bioneer, USA). 1 μg of RNA was mixed with 0.5 μg Oligo(dT)18 Primer (Fermentas, USA) and it was added to the kit, then reached 20 μl using diethyl pyrocarbonate (DEPC)-treated water. The kit was incubated at 42°C for 60 min and finally at 70°C for 10 min to deactivate reverse transcriptase enzyme.
8
Reverse Transcription and EMSA for STAT3
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Total RNA from YD-38 cells were prepared using RNeasy Mini kit (Quiagen, CA, USA) according to the manufacturer's instructions. Equal amount of RNA from each sample reverse transcribed using AccuPower RT-premix kit (Bioneer, Korea) and oligo(dT) primers. PCR was performed using 2 µl of the reverse transcription product. The PCR reactions were performed in 25-30 cycles of denaturation 94-95˚C, annealing 56-60˚C and an extension of 72˚C. The primers used for the amplification are listed in the Table I. After amplification, the products were visualized in 1.2% agarose containing ethidium bromide.
Electrophoretic mobility shift assay (EMSA). STAT3 DNA binding activity was detected using EMSA (19) . Gingival cancer cells were grown to ~80% confluence and nuclear protein extracts were prepared using the Nuclear Extraction kit (Affymetrix, CA, USA). EMSA was performed with EMSA gel shift kit (Panomics) according to the manufacturer's protocol. Briefly, the nuclear proteins prepared were subjected for hybridization with a double-stranded, biotin-labeled oligonucleotide probe containing the consensus-binding site for STAT3 (sense strand, 5'-CATGTTATGCATATTCCTGTAAGTG-3'). The protein-DNA complexes were resolved in a 6% non-denaturing PAGE gel and transferred to Pall Biodyne B nylon membrane (Pall Life Sciences, NY, USA) and detected using streptavidin-HRP and a chemiluminescent substrate.
Electrophoretic mobility shift assay (EMSA). STAT3 DNA binding activity was detected using EMSA (19) . Gingival cancer cells were grown to ~80% confluence and nuclear protein extracts were prepared using the Nuclear Extraction kit (Affymetrix, CA, USA). EMSA was performed with EMSA gel shift kit (Panomics) according to the manufacturer's protocol. Briefly, the nuclear proteins prepared were subjected for hybridization with a double-stranded, biotin-labeled oligonucleotide probe containing the consensus-binding site for STAT3 (sense strand, 5'-CATGTTATGCATATTCCTGTAAGTG-3'). The protein-DNA complexes were resolved in a 6% non-denaturing PAGE gel and transferred to Pall Biodyne B nylon membrane (Pall Life Sciences, NY, USA) and detected using streptavidin-HRP and a chemiluminescent substrate.
9
Cloning and Characterization of hRANKL
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The RNA used to clone hRANKL cDNA was extracted from MG63 cells (ATCC CRL-1427) expressing RANKL. The quality of the extracted RNA was verified by agarose gel electrophoresis. The cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea), according to the manufacturer’s instructions. Amplification and cloning of the hRANKL fragment were carried out in a reaction mixture comprising KOD polymerase buffer, 10 mM dNTPs, 25 mM magnesium chloride (MgCl2), 10 μM primers (hRANKL-BclI: 5′-TGATCAAAGCTTGAAGCTCAGCCTTTTGC-3′ and hRANKL-Xho I: 5′-CTCGAGATCTATATCTCGAACTTTAAAAGCCCC-3′), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA) and 2 μL of the RANKL gene construct (template). The thermal cycling conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 70 °C for 30 s. The polymerase chain reaction (PCR) product was cloned into the BamHI/XhoI sites of a pMX vector (CELL BIOLABS, USA). Sequence analyses were carried out using programs in Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA).
10
Melanoma Cell Total RNA Extraction and RT-PCR
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Total RNA was isolated from melanoma cells (1.0 × 104 cells/60 mm culture dish) using TRIzolTM Reagent (Thermo Fisher Scientific, Frederick, MD, USA). Subsequently, 1.0 µg of RNA was utilized for cDNA synthesis through the AccuPower® RT PreMix kit (Bioneer Corp., Daejeon, Republic of Korea). The cDNA for RT-PCR was generated by amplification with primers specific for β-actin, ST3 beta-galactoside alpha-2,3-sialyltransferase 5 (ST3GAL5), and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (ST8SIA1). The RT-PCR was conducted using a TaKaRa PCR Thermal Cycler Dice Gradient System (Takara, Dalian, China). Detailed primer information and the specific PCR conditions employed in this study are provided in Table S1 .
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