Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA,USA) according to the manufacturer's instructions. To obtain cDNA, total RNA (1 μg) was reverse-transcribed using an oligo(dT)16 primer. The cDNA was amplified using a high capacity cDNA synthesis kit (Bioneer, Daejon, Korea) with a thermal cycler (Bio-rad, Hercules, CA, USA). Real-time polymerase chain reaction (PCR) was performed with STEP ONE (Applied Biosystems, Foster City, CA, USA) using a SYBR green premix according to the manufacturer's instructions (Applied Biosystems). Primers were synthesized by Bioneer. The following primer sequences were used: mouse SREBP-1 5′- GAGGCCAAGCTTTGGACCTGG-3′ (sense) and 5′- CCTGCCTTCAGGCTTCTCAGG-3′ (antisense); mouse FAS 5′- ATTGCATCAAGCAAGTGCAG-3′ (sense) and 5′- GAGCCGTCAAACAGGAAGAG-3′ (antisense); mouse ACC 5′- TGAAGGGCTACCTCTAATG-3′ (sense) and 5′- TCACAACCCAAGAACCAC-3′ (antisense); mouse PPARα 5′- CTGCAGAGCAACCATCCAGAT-3′ (sense) and 5′- GCCGAAGGTCCACCATTTT -3′ (antisense); and mouse Sirt1 5′-ATCGGCTACCGAGACAAC-3′ (sense) and 5′- GTCACTAGAGCTGGCGTGT-3′ (antisense). The relative level of PCR products was determined on the basis of the threshold cycle value. Glyceraldehyde-3-phosphate dehydrogenase was used as a reference gene for normalization. Melting curve analysis was done after amplification to verify the accuracy of the amplicon.
High capacity cdna synthesis kit
Manufactured by Bioneer
The High-capacity cDNA synthesis kit is a laboratory tool designed for the efficient conversion of RNA to complementary DNA (cDNA). It provides a streamlined process for generating high-quality cDNA from various RNA sources, enabling downstream applications such as gene expression analysis, cloning, and sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Thermal cycler, by Bio-Rad (7 mentions)
Sybr green premix, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Stepone, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Pcr premix, by Bioneer (3 mentions)
Ethidium bromide, by Merck Group (3 mentions)
Gel documentation system, by Fujifilm (3 mentions)
Pcr machine, by Bio-Rad (2 mentions)
Stepone model, by Thermo Fisher Scientific (1 mentions)
9 protocols using high capacity cdna synthesis kit
1
Quantitative Real-Time PCR for Gene Expression
2
RNA Extraction and RT-qPCR Analysis of Murine Melanogenic Genes
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Total RNA was extracted using TRIzol reagent (Invitrogen), in accordance with the manufacturer’s instructions. To obtain cDNA, total RNA (2 µg) was reverse-transcribed using an oligo(dT) primer. The cDNA was amplified using the High-Capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) in a PCR machine (Bio-Rad, Hercules, CA, USA). PCR was performed using a PCR premix (Bioneer), and real-time RT-PCR was performed using the StepOne model (Applied Biosystems, Foster City, CA, USA) and SYBR Green premix, according to the manufacturer’s instructions (Applied Biosystems). Primers were synthesized by Bioneer. The following primer sequences were used: mouse tyrosinase: 5′-ATAACAGCTCCCACCAGTGC-3′ (sense) and 5′-CCCAGAAGCCAATGCACCTA-3′ (antisense); mouse MITF: 5′-CTGTACTCTGAGCAGCAGGTG-3′ (sense) and 5′-CCCGTCTCTGGAAACTTGATCG-3′ (antisense); and mouse TRP-1: 5′-AGACGCTGCACTGCTGGTCAAGCCTGTAGCCCACGTCGTA-3′ (sense) and 5′-GCTGCAGGAGCCTTCTTTCT-3′ (antisense). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control for qRT-PCR [30 (link)].
3
qRT-PCR Analysis of Melanogenesis Genes
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Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. To obtain cDNA, the total RNA (2 μg) was reverse transcribed using an oligo (dT) 16 primer. The cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejon, Korea) with a thermal cycler (BioRad, Hercules, CA, USA). Subsequently, PCR was performed using a PCR premix (Bioneer, Daejon, Korea), and real-time RT-PCR was performed with STEP ONE (Applied Biosystems, Foster City, CA, USA) using a SYBR Green premix in accordance with the manufacturer’s instructions (Applied Biosystems). Primers were synthesized by Bioneer. The following primer sequences were used: mouse TYR, 5′-ATAACAGCTCCCACCAGTGC-3′ (sense) and 5′-CCCAGAAGCCAATGCACCTA-3′ (antisense); mouse MITF, 5′-CTGTACTCTGAGCAGCAGGTG-3′ (sense) and 5′-CCCGTCTCTGGAAACTTGATCG-3′ (antisense); mouse TRP-1, 5′-AGACGCTGCACTGCTGGTCAAGCCTGTAGCCCACGTCGTA-3′ (sense) and 5′-GCTGCAGGAGCCTTCTTTCT-3′ (antisense). The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for the quantitative RT-PCR experiments.
4
Transcriptome Analysis by RT-PCR
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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To obtain cDNA, total RNA (2 µg) was reverse-transcribed using an oligo(dT)16 primer. The cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) and a thermal cycler (Bio-Rad, Hercules, CA, USA). Real-time PCR (RT-PCR) was performed with STEP ONE (Applied Biosystems, Foster City, CA, USA) using SYBR Green premix according to the manufacturer’s instructions (Applied Biosystems). Primers were synthesized by Bioneer (Table 2 ).
5
RT-PCR Analysis of Parkin Gene Expression
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Total RNA was extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA (2 µg) was reverse transcribed using oligo (dT)16 primers to obtain cDNA. The cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Republic of Korea) with a thermal cycler (Bio-Rad, Hercules, CA, USA). The amplified products were separated on a 2% agarose gel, stained with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA), and visualized with a gel documentation system (Fujifilm, Tokyo, Japan). The primer sequences were as follows: human parkin sense 5′-TCCTTCCTGCTGTCAGTGTG-3′, and antisense 5′-GCAGAGACCGTGGAGAAAAG-3′; human GAPDH sense 5′-GAAGGTGAAGGTCGGAGTC-3′, and antisense 5′-GAAGATGGTGATGGGATTTC-3′. Equal loading was verified based on the GAPDH level.
6
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. To obtain cDNA, total RNA (2 µg) was reverse-transcribed using an oligo primer (dT). The cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) with a PCR machine (Bio-Rad, Hercules, CA, USA). Subsequently, PCR was performed using a PCR premix (Bioneer), and real-time RT-PCR was performed with STEP ONE (Applied Biosystems, Foster City, CA, USA) using a SYBR Green premix in accordance with the manufacturer’s instructions (Applied Biosystems). Primers were synthesized by Bioneer. The following primer sequences were used: mouse tyrosinase 5′-ATAACAGCTCCCACCAGTGC-3′ (sense) and 5′-CCCAGAAGCCAATGCACCTA-3′ (antisense); mouse MITF, 5′-CTGTACTCTGAGCAGCAGGTG-3′ (sense) and 5′-CCCGTCTCTGGAAACTTGATCG-3′ (antisense); mouse TRP-1 5′-AGACGCTGCACTGCTGGTC AAGCCTGTAGCCCACGTCGTA-3′ (sense) and 5′-GCTGCAGGAGCCTTCTTTCT-3′ (antisense). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control for qRT-PCR experiments [46 (link)].
7
Quantitative Analysis of Fibrosis Markers
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Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. To obtain cDNA, total RNA (2 µg) was reverse-transcribed using oligo(dT)16 primer. The cDNA obtained was amplified with a high-capacity cDNA synthesis kit (Bioneer, Daejon, Korea) using a thermal cycler (Bio-Rad, Hercules, CA, USA). Real-time PCR was performed with STEP ONE (Applied Biosystems, Foster City, CA, USA) using SYBR green premix according to the manufacturer’s instructions (Applied Biosystems). Primers were synthesized by Bioneer. The following primer sequences were used: human α-SMA 5’-CGCATCCTCATCCTCCCT-3’ (sense) and 5’-GGCCGTGATCTCCTTCTG-3’ (antisense); human PAI-1 5’-CGCCAGAGCAGGACGAA-3’ (sense) and 5’-CATCTGCATCCTGAAGTTCTCA-3’ (antisense); human Col 1A1 5’-CCTGGGTTTCAGAGACAACTTC-3’ (sense); mouse α-SMA 5’-TCCTCCCTGGAGAAGAGCTAC-3’ (sense) and 5’-TATAGGTGGTTTCGTGGATGC-3’ (antisense); mouse PAI-1 5’-GACACCCTCAGCATGTTCATC-3’ (sense) and 5’-AGGGTTGCACTAAACATGTCAG-3’ (antisense); mouse Col 1A1 5’-ACCTGTGTGTTCCCTACTCA-3’ (sense) and 5’-GACTGTTGCCTTCGCCTCTG-3’ (antisense); and 5’-TCCACATGCTTTATTCCAGCAATC-3’ (antisense). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for RT-PCR.
8
RT-PCR Analysis of ECM Markers
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TRIzol (Invitrogen) was used to obtain total RNA extracts according to the manufacturer’s protocol. To synthesize cDNA, total RNA (2 μg) was reverse-transcribed using an oligo-dT18 primer. The synthesized cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) with a thermal cycler (Bio-Rad, Hercules, CA, USA). PCR-amplified products were separated by 2% agarose gel electrophoresis and staining with ethidium bromide (Sigma, St. Louis) and imaged using a gel documentation system (Fujifilm). The following PCR primer sequences were used: human MMP3 sense 5′-AACCTGTCCCTCCAGAACCT-3′ and antisense 5′-GGAAGAGATGGCCAAAATGA-3′; human MMP9 sense 5′-CTCGAACTTTGACAGCGACA-3′ and antisense 5′-GCCATTCACGTCGTCCTTAT-3′; human Col1A1 sense 5′-CACAGAGGTTTCAGTGGTTTGG-3′ and antisense 5′-GCACCAGTAGCACCATCATTTC-3′; human GADD153/CHOP sense 5′-AGGGAGAACCAGGAAACGGAAACA-3′ and antisense 5′-TCCTGCTTGAGCCGTTCATTCTCT-3′; human and human GAPDH sense 5′-GAAGGTGAAGGTCGGAGTC-3′ and antisense 5′-GAAGATGGTGATGGGATTTC-3′. GAPDH was used as a reference gene for normalization.
9
Melanogenesis Gene Expression Analysis
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TRIzol (Invitrogen) was used to obtain the total RNA extract according to the manufacturer’s protocol. To synthesize cDNA, total RNA (2 µg) was reverse-transcribed using oligo dT18 primer. The synthesized cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) with a thermal cycler (Bio-Rad, Hercules, CA, USA). PCR-amplified products were separated using 2% agarose gel, including ethidium bromide (Sigma, St. Louis, MO, USA), and imaged in a gel documentation system (Fujifilm, Tokyo, Japan) [30 (link)]. The following primer sequences were used: mouse tyrosinase 5′-ATAACAGCTCCCACCAGTGC-3′ (sense) and 5′-CCCAGAAGCCAATGCACCTA-3′ (antisense) (NM_011661.5); mouse MITF, 5′-CTGTACTCTGAGCAGCAGGTG-3′ (sense) and 5′-CCCGTCTCTGGAAACTTGATCG-3′ (antisense) (NM_001178049.1); mouse TRP-1 5′-AGACGCTGCACTGCTGGTC AAGCCTGTAGCCCACGTCGTA-3′ (sense) and 5′-GCTGCAGGAGCCTTCTTTCT-3′ (antisense) (NM_001282015.1). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control for qRT-PCR experiments [20 (link)].
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