The gene expression of four genes selected from RNA sequencing and related publications was examined by RT‐PCR. A549 cells were treated with 6 μM of S4‐2‐2 or DMSO for 15 h, and total mRNA was isolated with TRIzol according to a standard protocol. An m‐MLV reverse transcriptase kit (Bioneer Co.) was used to synthesize cDNA from 2 μg of mRNA. The sequences of the primers for the genes are listed in Table S3 , and the human GAPDH gene was used as an internal control.
M mlv reverse transcriptase kit
Manufactured by Bioneer
The M-MLV reverse transcriptase kit is a laboratory tool used for the conversion of RNA into complementary DNA (cDNA). The kit contains the M-MLV reverse transcriptase enzyme, which catalyzes the reverse transcription process. This process is essential for various molecular biology applications, such as gene expression analysis and cDNA library construction.
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Lab products found in correlation
Accupower 2x greenstar qpcr master mix, by Bioneer (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Desired primers, by Bioneer (3 mentions)
Rotor gene 3000, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Geneamp pcr system 2700, by Thermo Fisher Scientific (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (2 mentions)
Easy blue total rna extraction kit, by iNtRON Biotechnology (2 mentions)
Riboex total rna reagent, by GeneAll (1 mentions)
Fluorometric thermal cycler, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rotor gene q thermocycler, by Qiagen (1 mentions)
Trizol reagent, by GeneAll (1 mentions)
14 protocols using m mlv reverse transcriptase kit
1
Transcriptional Profiling of Targeted Genes
2
RNA Isolation and qPCR Analysis
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RiboEx Total RNA reagent (Geneall biotechnology, Seoul, Korea) was used for RNA isolation from liver, muscle, and adipose tissue. cDNA was synthesized using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase kit (Bioneer, Daejeon, Korea). The Rotor-Gene 3000 (Corbett Research, Sydney, Australia) was utilized to conduct quantitative polymerase chain reaction (qPCR) by using the AccuPower 2X Greenstar qPCR Master Mix (Bioneer, Daejeon, Korea). Gene expression was normalized using β-actin, a housekeeping gene and expressed as fold change of the HF group followed by the 2 − ∆∆Ct method [29 (link)]. Primer sequences used for this study are listed in Table S2 .
3
Quantitative Analysis of Gene Expression in Dermal Fibroblasts
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Human dermal fibroblast (HDF) cells were seeded in a 6-well plate at a density of 80,000 per well. After a 3-day treatment, 250 µL of the TRIzol reagent (Invitrogen, Waltham, MA, USA) was added to each well for total RNA extraction. Subsequently, 1 µg of the total RNA, along with the M-MLV Reverse Transcriptase kit (Bioneer, Daejeon, Republic of Korea), were used for cDNA synthesis in a 20 µL reaction, which was later diluted to 100 µL and utilized as a template in the following real-time qPCR reactions. Real-time qPCR was conducted using the AccuPower® 2X GreenStar™ qPCR Master Mix (Bioneer), and all reactions were executed in triplicate with the expression levels of the markers normalized to the expression level of 18s ribosomal RNA.
4
Quantitative Real-Time PCR Analysis of RNA
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Total RNA was isolated from muscle tissues and C2C12 muscle cells using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). A total of 1 μg of RNA was subsequently reverse-transcribed to cDNA using a MMLV reverse transcriptase kit (Bioneer, Daejeon, Korea) following the manufacturer’s instructions and the reaction was performed at 37 °C for 60 min by incubation at 95 °C for 5 min with the use of GeneAMP® PCR system 2700 (Applied Biosystems, Foster City, CA, USA). The polymerase chain reaction (PCR) was performed using an AccuPower® 2X GreenStar™ qPCR Master Mix (Bioneer) and a fluorometric thermal cycler (Corbett Research, NSW, Australia). The conditions used were as follows: pre-denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation (95 °C, 15 s), annealing (60 °C, 20 s), and extension (72 °C, 20 s). Primers used are shown in Supplementary Tables S1 and S2 . Results were relatively quantified using the ΔΔCt method and expressed as the fold change compared to NOR group or C2C12 vehicle control [32 (link)].
5
Zebrafish Transcriptome Analysis with FO and GP
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Zebrafish larvae at 3 dpf were treated with 0–100 µg/mL FO or 4 mM GP, and total RNA was extracted at 9 and 12 dpf using an easy-BLUETM total RNA extraction kit. The RNA was reverse-transcribed by an MMLV reverse transcriptase kit (BIONEER, Daejeon, Republic of Korea) and synthetic cDNA was amplified using following primers (Table 1 ).
6
Reverse Transcription and PCR Analysis
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions and reverse transcribed using an M-MLV reverse transcriptase kit (BioNEER, Daejeon, Korea) to produce cDNAs. The RT-generated cDNAs encoding iNOS, COX-2, TNF-α, and IL-1β genes were amplified by PCR using desired primers (BioNEER). Following amplification, the PCR products were separated on 1.5% agarose gel electrophoresis, stained with ethidium bromide (EtBr, Sigma-Aldrich Chemical Co.) and visualized under ultraviolet illumination. In a parallel experiment, glyceraldehyde 3-phosphate dehydrogenase (GPDH) was used as the internal control. The PCR primers were as follows: iNOS forward, 5′ATG TCC GAA GCA AAC ATCAC3′ and reverse, 5′TAA TGT CCA GGA AGT AGG TG3′; COX-2 forward, 5′-CAG CAA ATC CTT GCT GTT CC-3′ and reverse 5′-TGG GCA AAG AAT GCA AAC ATC-3′, TNFα forward, 5′TCT CAT CAG TTC TAT GGC CC3′ and reverse, 5′GGG AGT AGA CAA GGT ACA AC3′; IL1β forward, 5′GGG CTG CTT CCA AAC CTT TG3′ and reverse, 5′GCT TGG GAT CCA CAC TCT CC3′; and GAPDH forward, 5′AGG CCG GTG CTG AGT ATG TC3′ and reverse, 5′TGC CTG CTT CAC CAC CTT CT3′.
7
Quantifying mRNA Expression in eWAT
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Ten milligrams of eWAT was homogenized in 1 mL of TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted using the RNeasy Lipid Tissue Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany) and quantified by the NanoDrop1000 spectrophotometer (Thermo Scientific). The Moloney Murine Leukemia Virus (MMLV) Reverse Transcriptase Kit (Bioneer, Daejeon, Korea) was used to generate cDNA from 1 μg of RNA. RT-qPCR was performed using AccuPower 2X Greenstar qPCR Master Mix (Bioneer) on a Rotor-Gene Q thermocycler (Qiagen). Relative mRNA expression was normalized to that of β-actin as an internal control, calculated by the 2−∆∆Ct method [58 (link)], and was expressed as the fold difference related to the HF group. Primers sequences used in RT-qPCR are presented in Table S1 .
8
Quantification of Gene and microRNA Expression in Adipose Tissue
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Total RNA was extracted from epididymal adipose tissue using TRIzol reagent (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. cDNA was synthesized from total RNA using a Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase kit (Bioneer Co., Daejeon, Korea). Real-time qPCR was performed using Rotor Gene 3000 (Corbett Research, Mortlake, N.S.W., Australia) and AccuPower 2X Greenstar qPCR MasterMix (Bioneer Co., Daejon, Korea). Primers used for real-time qPCR analysis are described in Supplementary Table S2 . Data analysis was conducted by the 2−ΔΔCt method. β-actin was used as the reference gene for normalization.
For the analysis of miR expression, cDNA was synthesized using a miRNA cDNA Synthesis Kit with Poly (A) Polymerase Tailing (ABM Inc., Richmond, BC, Canada). The synthesized cDNA was amplified using the EvaGreen miRNA qPCR Master Mix (ABM Inc.). Quantification of miRs was carried out using miR-21, miR-132, and U6 specific primers (ABM Inc). Real-time qPCR amplification was performed using the Rotor Gene 3000 (Corbett Research). Levels of miR-21 and miR-132 were normalized to U6 snRNA and determined using the 2−ΔΔCt method.
For the analysis of miR expression, cDNA was synthesized using a miRNA cDNA Synthesis Kit with Poly (A) Polymerase Tailing (ABM Inc., Richmond, BC, Canada). The synthesized cDNA was amplified using the EvaGreen miRNA qPCR Master Mix (ABM Inc.). Quantification of miRs was carried out using miR-21, miR-132, and U6 specific primers (ABM Inc). Real-time qPCR amplification was performed using the Rotor Gene 3000 (Corbett Research). Levels of miR-21 and miR-132 were normalized to U6 snRNA and determined using the 2−ΔΔCt method.
9
Quantitative RT-PCR Analysis of mRNA Expression
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Total RNA was isolated using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. Isolated RNA was subsequently reverse-transcribed to cDNA using a MMLV Reverse Transcriptase Kit (Bioneer, Daejeon, Korea) and incubation at 37 °C for 60 min followed by incubation at 95 °C for 5 min using GeneAMP® PCR system 2700 (Applied Biosystems, Foster City, CA, USA). mRNA expression was determined by qRT-PCR as described previously [38 (link)]. Primer sequences used are provided in Table S1 . mRNA level of each target gene was normalized to the housekeeping gene, β-actin and expressed as the fold change relative to C2C12 control cells.
10
Epididymal Adipose Tissue RNA Isolation
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Isolation of RNA from epididymal adipose tissue was performed using an RNeasy Lipid Tissue Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized from 1 μg of total RNA using a MMLV Reverse Transcriptase Kit (Bioneer, Daejeon, Korea). The reaction was performed at 37 °C for 60 min followed by incubation at 95 °C for 5 min. Primers used are shown in Table 2 . The polymerase chain reaction (PCR) parameters were as follows: pre-denaturation at 95 °C for 10 min, followed by 50 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s and extension at 72 °C for 20 s. Data were analyzed using the ∆∆Ct method for relative quantification [44 (link)]. Expression of each target was normalized to the average of β-actin as a control and expressed as the fold change related to the NOR group.
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