Cells were collected 24 h after sensitization with inducers. RNA was isolated by using TRIZOL reagent, according to the manufacture’s guidelines (Invitrogen Life Technologies, Carlsbad, CA, USA). An equal amount of RNA (3 µg) was reverse transcribed using a PCR premix (Bioneer Co., Daejoon, Korea), 0.5 µg/mL oligo-(dT)15 primer, and 0.5 µg/mL hexamer primer. The cDNA was resynthesized at 42 °C for 60 min and was incubated at 94 °C for 5 min to stop the reaction. PCR amplification was performed using a PCR premix (Bioneer) and designated primer pairs outlined in Supplementary Materials Table S2 . Amplified products were observed by gel electrophoresis and detected by nucleic acid staining (NobleBio Inc., Gyeonggi, Korea) under UV illumination. GAPDH was used for normalization.
Pcr premix
Manufactured by Bioneer
Sourced in United States
PCR premix is a ready-to-use solution containing all the necessary components for performing polymerase chain reaction (PCR) experiments. It includes a thermostable DNA polymerase, buffer, dNTPs, and other reagents required for DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Trizol, by Thermo Fisher Scientific (10 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (6 mentions)
Rt premix, by Bioneer (6 mentions)
Pcr machine, by Thermo Fisher Scientific (3 mentions)
Reverse transcriptase premix, by Bioneer (3 mentions)
Oligo dt, by Bioneer (3 mentions)
High capacity cdna synthesis kit, by Bioneer (3 mentions)
Sybr green premix, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Oligo dt 15 dimer, by Bioneer (3 mentions)
Milli q purification system, by Merck Group (2 mentions)
Oligonucleotides, by Bioneer (2 mentions)
Dna extraction kit, by Bioneer (2 mentions)
Ecodry premix, by Takara Bio (2 mentions)
Imagequant las 500, by GE Healthcare (2 mentions)
Pcr machine, by Bio-Rad (2 mentions)
Stepone, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
58 protocols using pcr premix
1
Transcriptional Analysis of Induced Cells
2
RNA Extraction and Real-Time PCR Analysis
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Total RNA was extracted using a PureLink RNA Mini Kit (Ambion) according to the manufacturer’s instructions. Reverse transcription was performed with Accupower RT-PCR premix (Bioneer, Sung Nam, Republic of Korea) according to the manufacturer’s protocol. cDNA, primers, and diethyl pyrocabonate (DEPC) were combined with a PCR premix (Bioneer) for PCR analysis, and PCR products were loaded on 1.5% agarose gels with gel red (Koma, Seoul, Republic of Korea) and detected using a Bio-Rad Gel Doc XR system (Bio-Rad, Hercules, CA, USA). Quantitative real-time SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used for real-time PCR. The relative expression of all individual genes was calculated using the 2-ΔΔCt method and normalized to the endogenous expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems, Foster City, CA, USA). For detection of SOX2 transcripts, we used a Total-SOX2 primer binding to the Coding DNA Sequence (CDS) and an Endo-SOX2 primer binding to the SOX2 3′-untranslated region (UTR), which does not exist in the SOX2 mRNA. All primers are listed in Supplementary Table 1.
3
Quantification of cytokine gene expression in HaCaT cells
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Total cellular RNA was extracted from HaCaT cells using TRIzol (Invitrogen Co., Grand Island, USA) and the subsequent steps were described in a previous study [50 (link)]. Briefly, the concentration of RNA samples was quantified and the reverse transcriptase reaction was carried out for 60 min at 42 °C, followed by 5 min at 94 °C. A PCR premix (Bioneer, Korea) and a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA) were used for polymerase chain reaction (PCR) amplification. Primers for IL-8, MDC, RANTES, TARC, GAPDH were provided in Table S1 . PCR products were separated by 2.0% agarose gel electrophoresis and visualized with ethidium bromide staining under UV illumination. The density of PCR products was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
4
Fungal ITS Region Amplification
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The internal transcribed spacer region of ribosomal DNA (ITS) was amplified by using ITS primer ITS1-F (TCC GTA GGT GAA CCT GCG G) and ITS4-R (TCC TCC GCT TAT TGA TAT GC) [27] (Bioneer Co, Korea). Total 20 μL volume of PCR reaction mix were amplified using a thermo-cycler model (My TM Genie 32 Thermal Block-Bioneer, South Korea). The PCR reaction was performed in 20 total volumes consisting of 5 μL of PCR PreMix (Bioneer Corporation, South Korea), and 5 μL of DNA (50 ng) template, and 3μL of primer ITS1-F, and 3 μL ITS4-R, and 4μL of PCR demonized water. The following program settings were used: initial denaturation (95°C, 2 min), 35 cycles of denaturation 94°C for 30s, annealing (55°C, 1 min) and extension (72°C, 1 min), final extension phase was performed at 72°C during 10 min.
The PCR final products were loaded in a 1.5% agarose gel with Ethidium-bromide dye. DNA marker 100 bp was used (Bioneer Co., South Korea). Samples of 2μL mix with 2μL of 6X loading buffer and loaded in to the gel. Electrophoresis (model Bioneer Co., South Korea) was run at 80 V for 2 h. The DNA bands were visualized and photographed using special UV camera (model AE-9000 E-Graph Atto-Jaban).
The PCR final products were loaded in a 1.5% agarose gel with Ethidium-bromide dye. DNA marker 100 bp was used (Bioneer Co., South Korea). Samples of 2μL mix with 2μL of 6X loading buffer and loaded in to the gel. Electrophoresis (model Bioneer Co., South Korea) was run at 80 V for 2 h. The DNA bands were visualized and photographed using special UV camera (model AE-9000 E-Graph Atto-Jaban).
5
Transcriptional Regulation After UVB Exposure
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RNA was isolated from NHDFs treated with PVE (1, 10, 100 μg/mL) for 24 hours following UVB irradiation according to the manufacturer's instructions using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Polymerase chain reaction (PCR) amplification of the cDNA template was performed using PCR premix (Bioneer) and the following primer pairs: MMP-1, forward 5′-TGC GCA CAA ATC CCT TCT-3′, reverse 5′-TTC AAG CCC ATT TGG CAG TT-3′; MMP-3, forward 5′-GGCCAGGGATTAATGGAGAT-3′, reverse 5′-GGAACCGAGTCAGGTCTGTG-3′. Procollagen type I, forward 5′-CTC GAG GTG GAC ACC CT-3′, reverse 5′-CAG CTG GAT GGC CAC ATC GG-3′; TNF-α, forward 5′-AGGGGAAATGAGAGACGCAA-3′, reverse 5′-TTCCCCATCTCTTGCCACAT-3′; IL-6, forward 5′-CTCCTTCTCCACAAGCGCC-3′, reverse 5′-GCCGAAGAGCCCTCAGGC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5′-ACC ACA GTC CAT GCC ATC AC-3′, reverse 5′-CCA CCA CCC TGT TGC TGT AG-3′. Reverse transcription–PCR (RT-PCR) was performed in a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA) as previously described.14 (link) PCR products were stained with nucleic acid staining solution and separated by 2.0% agarose gel. Each experiment was repeated at least three times.
6
RNA Extraction and RT-PCR Analysis of Cultured Cells
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Cultured cells in conditioned media (containing 0, 100, 500, and 1000 μg/mL of TAO) were lysed immediately with a RibospinTM kit (GeneAll Biotech, Seoul, Korea) to extract total RNA. Quantification and quality assessment of RNA was undertaken with a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Isolated RNA was converted to cDNA using CycleScript RT Premix (dT20) (Bioneer, Dajeon, Korea), and cDNA was amplified using conventional PCR (PCR PreMix, Bioneer, Dajeon, Korea) under the following conditions: denaturation steps for 15 min at 95 °C, 40 amplification cycles (95 °C for 10 s, 59 °C for 15 s, 72 °C for 30 s) for annealing, a melting curve, and a final cooling step [40 (link)]. The sequences of the PCR primers used are provided in Table 1 , and the products were analyzed using iBright (FL1000, Thermo Fisher Scientific, Waltham, MA, USA) and iBright Analysis software 3.1.3 (Thermo Fisher Scientific, Waltham, MA, USA).
7
Quantifying Hepatic Cholesterol Metabolism Genes
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For the mRNA expression of SREBP-2, ACAT-2, and HMG-CoA, total RNA was extracted from liver tissue with TRIZOL solution using a homogenizer as previously described [19 (link)]. The RNA (2 μg) was annealed with OligodT by heating at 70°C for 10 min and kept in ice for cooling for another 10 min. The reverse transcriptase reaction was carried out using a commercially available premix (Bioneer, Daejeon, Republic of Korea) by heating at 42°C for 1.5 h, and the reaction was stopped by heating at 95°C for 5 min. The resulting cDNA was added to the PCR premix (Bioneer, Daejeon, Republic of Korea) with the respective target gene primers. The PCR product was run on 1% agarose gel stained with ethidium bromide (EtBr), and the gel images were developed using an imaging system (GE Healthcare, Chicago, IL, USA). The band intensities were normalized against GAPDH, which was used as the housekeeping gene. Similarly, after reverse transcriptase, the resultant cDNA was then added with SYBR Green (Thermo Fisher, Waltham, MA, USA) with primers of GAPDH, ACAT-1, and ACAT-2 for real-time PCR analysis using CFX96 Real Time System (Biorad, Hercules, CA, USA) as previously described [19 (link)].
8
Mycobacterium Identification Protocol
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Chemicals were procured from Sigma-Aldrich Chemicals, Korea. All the oligonucleotides, PCR pre-mix, and DNA extraction kits were purchased from Bioneer, Korea. All washing solvents for the substrates are of HPLC grade from SK Chemicals, Seoul, Korea. Ultrapure water (18 M Ω/cm) was obtained from a Milli-Q purification system (Millipore). The 9G DNAChips were obtained from Biometrix Technology Inc., Chuncheon, Korea. The standard samples of Mycobacterium Tuberculosis (MTB) and NTM genotypes were obtained from the Korean Institute of Tuberculosis. Mycobacterium organism strains used: Mycobacterium Tuberculosis (H37RV); Mycobacterium Avium (M. Avium); Mycobacterium Abscessus (M. Abscessus), Mycobacterium Chelonae (M. Chelonae), and Mycobacterium Kansasii (M. kansasii).
9
Macrophage Gene Expression Analysis
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Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from WelGene (Daegu, Korea). Streptomycin and penicillin were obtained from Lonza (MD, USA). TRI reagent solution (AM9738) and SYBER green master mix were obtained from Applied Biosystems/Ambion (Warrington, UK). Oligo(dT) primers, RT premix, and PCR premix were obtained from Bioneer Co. (Daejeon, Korea). iNOS, COX-2, TNF-α, IL-1β, IL-6, Leptin, adiponectin, RGS5, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were obtained from Bioneer Co. (Table 1 ) (Daejeon, Korea). Total protein lysis buffer (PRO-PREP) and the PRO-Measure protein assay kit were obtained from iNtRON Biotechnology (Seoul, Korea). LPS (Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma (St. Louis, MO, USA). All other reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA).
10
cDNA Synthesis and Mouse NDP52 Gene Amplification
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For the synthesis of cDNA, total RNA was isolated using Trizol (Invitrogen) according to the manufacture’s protocol. cDNA was synthesized using 2 µg of RNA with EcoDry premix (Clontech, 639543). Mouse NDP52 gene was amplified using the PCR premix (Bioneer, Korea) following addition of 1 µL of cDNA product and 1 µM of primers (forward and reverse, Supplementary Table 6 ) on a PCR machine (Applied Biosystems). The reaction tubes were incubated at 96°C for 5 min, followed by 35 cycles of 96°C for 30 s, 58°C for 30 s and 72°C for 45 s. The PCR products were analyzed by the agarose gel (1.2%) electrophoresis.
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