Myod1

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

MyoD1 is a DNA-binding protein that plays a key role in the regulation of muscle-specific gene expression. It is a member of the MyoD family of transcription factors and is involved in the determination and differentiation of skeletal muscle cells.

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5 protocols using myod1

Quantitative Analysis of Myogenic Markers

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C2C12 cells were washed with ice-cold PBS and total RNA was extracted using the TRIzol reagent (Life Technologies). Digestion of contaminating genomic DNA was performed with DNA-free DNase treatment (Ambion). First strand cDNA was generated using a kit with random primers (High Capacity cDNA Reverse Transcription kit, Applied Biosystems) from 1 μg of total RNA. Newly synthesized cDNA was diluted 5-fold in DNA-free water and 5% of this cDNA was used in each real-time PCR assay, using a 7500 Real Time PCR System (Applied Biosystems). The standard curve method was used to calculate the relative mRNA levels for each transcript examined and actin beta (ACTB) was used as a reference to normalize the data. Specific assays used for the Taqman quantification were all from Applied Biosystems: MYOG: Mm00446195_g1; MYOD1: Mm01203489_g1; PAX-7: Mm01354484_m1; SOCS3: Mm00545913_s1; ACTB: Mm00607939_s1.

Pelosi M., De Rossi M., Barberi L, & Musarò A. (2014). IL-6 Impairs Myogenic Differentiation by Downmodulation of p90RSK/eEF2 and mTOR/p70S6K Axes, without Affecting AKT Activity. BioMed Research International, 2014, 206026.

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Gene Expression Analysis of Myogenesis

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Total RNA was extracted using the kit Direct-zol^™ RNA Miniprep Plus (Zymo Research #R2072) following the manufacturer’s protocol. The cDNA was synthesized using 1 μg of the total RNA with the reverse transcriptase (Bioline, Sensi FAST Kit). Gene expression levels were measured by RT-PCR using the cDNA with the Sensi-Fast Hi-Rox Kit (Bioline #Bio-82,020). The target genes’ relative expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH Hs02786624-g1 20x Applied Biosystem). The expression of the following genes was analyzed PAX3 (Hs00240950), Myf5 (Hs00929416-g1), MyoD1(Hs02330075-g1), and Myogenin (Hs01072232-m1, Applied Biosystems).

Luzzi A., Wang F., Li S., Iacovino M, & Chou T.F. (2023). Skeletal muscle cell protein dysregulation highlights the pathogenesis mechanism of myopathy-associated p97/VCP R155H mutations. Frontiers in Neurology, 14, 1211635.

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Myogenic Differentiation of HEK293 Cells

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Cellular scaffolds (Human embryonic kidney cells; HEK 293, 2×10 6 cells/mL), exposed to culture medium or GET-MyoD (P21-mR-MyoD-8R) (60 µg/mL) for 3 days, were serially sliced into five parallel sections each 1 mm in diameter and snap frozen at -80 ºC. RNA was extracted using a Qiagen RNeasy mini kit (Qiagen, UK) and quantified using a Nanodrop spectrophotometer (ND-100 Thermo Scientific). This was followed by reverse transcription into cDNA using SuperScript™ III Reverse Transcriptase enzyme (Invitrogen, UK) according to manufacturer's instructions. Quantification of the gene expression was performed using rT-qPCR analysis (ΔΔC T ) using Taqman assays for human Actin-β, MyoD1, Myogenin, Myf5 and Desmin (Applied Biosystems, UK) as detailed Dixon et al. [26] .

Eltaher H.M., Yang J., Shakesheff K.M, & Dixon J.E. (2016). Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate. Acta biomaterialia, 41.

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Immunofluorescence Analysis of Myogenesis

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Immunofluorescence analysis was performed, as described previously^{3 (link)}, using the following antibodies: mouse monoclonal antibody against active β-catenin (Millipore), rabbit polyclonal antibodies against KAT2B (Cell Signaling Technology) and CBP (Cell Signaling Technology) and mouse monoclonal antibodies against myosin heavy chain (MYH) (Sigam-Aldrich) and MyoD1 (Thermofisher). Confocal images were obtained with a confocal microscope (C2, Nikon). Fluorescent images of MYH immunostaining were captured by an inverted fluorescence microscope (IX73, Olympus). A total of 10 fields, randomly selected from three independent experiments, were used for the quantification of the length of myotubes and fusion index.

Suzuki A., Minamide R, & Iwata J. (2018). The role of acetyltransferases for the temporal-specific accessibility of β-catenin to the myogenic gene locus. Scientific Reports, 8, 15057.

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Quantifying BSC Gene Expression

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To assess relative gene expression between BSCs cultured in various media types, qPCR was performed on proliferative (70% confluency) and differentiated cells from P3 following standard protocols. Briefly, RNA was harvested using an RNEasy Mini kit (Qiagen #74104, Hilden, Germany) and cDNA was prepared via the iScript cDNA synthesis kit (Bio-Rad #1708890, Hercules, CA, USA) using 1 μg of RNA for each reaction. Next, qPCR was performed using 2 μL of cDNA and 1x TaqMan Fast Universal PCR Master Mix without AmpErase UNG (ThermoFisher #4352042). Primers used in this study were: 18S (ThermoFisher #Hs03003631), Pax3 (ThermoFisher #Bt04303789), MyoD1 (ThermoFisher #Bt03244740), Myogenin (ThermoFisher #Bt03258929), and Myosin Heavy Chain (ThermoFisher #Bt03273061). Reactions were performed according to the manufacturer’s instructions on a Bio-Rad CFX96 Real Time System thermocycler, and results were analyzed as 2^−ΔΔct normalized to expression of the 18S housekeeping gene and analyzed relative to proliferative BSC-GM expression for each gene.

Stout A.J., Rittenberg M.L., Shub M., Saad M.K., Mirliani A.B., Dolgin J, & Kaplan D.L. (2023). A Beefy-R culture medium: replacing albumin with rapeseed protein isolates. Biomaterials, 296, 122092.

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