Transcriptional Analysis of Neuromuscular Genes

RNA from GCM was extracted using TRIzol (Invitrogen) and purified with PureLink RNA columns (Life Technologies). RNA samples were treated with DNase I, and reverse transcription was performed with High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Real-time PCR was performed using the TaqMan Gene expression assay (Applied Biosystems) following the manufacturer’s instructions, on cDNA specimens in triplicate, using 1× Universal PCR Master Mix (Life Technologies) and 1× mix containing specific receptors probes (Life Technologies). Relative quantification was calculated from the ratio between the cycle number (Ct) at which the signal crossed a threshold set within the logarithmic phase of the given gene and that of the reference β-actin gene (4310881E; Life Technologies). Mean values of the triplicate results for each animal were used as individual data for 2-ΔCt statistical analysis. The following is the list of probes used: nicotinic cholinergic receptor, gamma subunit (AChRγ) (CHRNG; Mm00437419_m1; Life Technologies), epsilon subunit (AChRε) (CHRNE; Mm00437411_m1; Life Technologies), alpha subunit (AChRα1) (CHRNA1; Mm00431629_m1; Life Technologies), beta subunit (AChRβ1) (CHRNB1; Mm00680412_m1; Life Technologies), delta subunit (AChRδ) (CHRND; Mm00445545_m1; Life Technologies), tumour necrosis factor alpha (TNF-alpha) (Mm00443258_m1; Life Technologies), insulin-like growth factor 1 (IGF1) (Mm00439560_m1; Life Technologies), CD4 (Mm00442754_m1; Life Technologies), forkhead box P3 (FOXP3) (Mm00475162_m1; Life Technologies), amphiregulin (Mm00437583_m1; Life Technologies), CD11c (Mm00498701_m1; Life Technologies), interferon-γ (IFNγ) (Mm01168134_m1; Life Technologies), transforming growth factor-β1 (Mm01178820_m1; Life Technologies), CD8a (Mm01182107_g1; Life Technologies), MyoD1 (Mm00440387_m1; Life Technologies), MyoG (Mm00446194_m1; Life Technologies), and Tmem8c (Mm00481256_m1; Life Technologies).

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Margotta C., Fabbrizio P., Ceccanti M., Cambieri C., Ruffolo G., D’Agostino J., Trolese M.C., Cifelli P., Alfano V., Laurini C., Scaricamazza S., Ferri A., Sorarù G., Palma E., Inghilleri M., Bendotti C, & Nardo G. (2023). Immune-mediated myogenesis and acetylcholine receptor clustering promote a slow disease progression in ALS mouse models. Inflammation and Regeneration, 43, 19.

Publication 2023

Actin Alpha subunit Amphiregulin Animal Assay Cdna Dnase i Gamma Gene Gene expression Insulin like growth factor 1 Interferon γ Myod1 Nicotinic cholinergic receptor Real time pcr Reverse transcription Subunit Transforming growth factor β1 Trizol Tumour necrosis factor alpha

Corresponding Organization :

Other organizations : Mario Negri Institute for Pharmacological Research, Sapienza University of Rome, Istituto Pasteur, University of L'Aquila, IRCCS Ospedale San Raffaele, Fondazione Santa Lucia, Istituti di Ricovero e Cura a Carattere Scientifico, Azienda Ospedaliera di Padova

Top 5 similar protocols

Variable analysis

independent variables

RNA extraction method (TRIzol)
RNA purification method (PureLink RNA columns)
DNase I treatment
Reverse transcription method (High-Capacity cDNA Reverse Transcription Kit)
Real-time PCR method (TaqMan Gene expression assay)

dependent variables

Relative quantification of gene expression for the following targets:
Nicotinic cholinergic receptor, gamma subunit (AChRγ)
Epsilon subunit (AChRε)
Alpha subunit (AChRα1)
Beta subunit (AChRβ1)
Delta subunit (AChRδ)
Tumour necrosis factor alpha (TNF-alpha)
Insulin-like growth factor 1 (IGF1)
Forkhead box P3 (FOXP3)
Amphiregulin
CD11c
Interferon-γ (IFNγ)
Transforming growth factor-β1
MyoD1
Tmem8c

control variables

Endogenous control: β-actin gene

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