Total RNA was extracted from tissues and cells by using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA was reverse-transcribed by using a PrimeScript RT reagent kit (Takara Bio Inc.), and small RNA polyadenylation was performed (Zhang et al., 2012 (link)). Real-time PCR was performed on a real-time PCR system (ABI 7900; Applied Biosystems). The primer sequences are provided in Table S3 . Mouse monoclonal anti–TEF-1 (610922; BD), rabbit polyclonal anti-MyoD (sc-760; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-MEF2 (sc-313; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-myogenin (sc-12732; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti–α-tubulin (T6199; Sigma-Aldrich), mouse monoclonal anti–β-actin (A5316; Sigma-Aldrich), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH; KC-5G4; KangChen Bio-tech Inc.), rabbit polyclonal anti-TEAD2 (21159-1-AP [Proteintech]; 33900 [Signalway Antibody]), rabbit polyclonal anti-TEAD3 (13120-1-AP; Proteintech), and rabbit polyclonal anti-TEAD4 (12418-1-AP) antibodies were used for Western blot analysis.
Protocol detail
RNA Extraction and Real-Time PCR Analysis
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Trizol reagent
PrimeScript RT reagent kit
ABI 7900
anti-MyoD (sc-760;
(sc-313;
mouse monoclonal anti–α-tubulin
mouse monoclonal anti–β-actin (A5316;
Actin
Antibodies
Antibody
Cells
Gapdh
Glyceraldehyde 3 phosphate dehydrogenase
Mouse
Myogenin
Primer
Rabbit
Real time pcr
Rna polyadenylation
Tissues
Trizol
Western blot analysis
α tubulin
Corresponding Organization : Center for Excellence in Molecular Cell Science
Other organizations : University of Chinese Academy of Sciences, Children's Hospital of Fudan University, Shanghai Xuhui Central Hospital, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan Hospital, Fudan University, Sun Yat-sen University
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Variable analysis
independent variables
- Total RNA extraction by Trizol reagent, Reverse transcription by PrimeScript RT reagent kit, and Small RNA polyadenylation
- Gene expression levels measured by Real-time PCR
- Primer sequences provided in Table S3, Mouse monoclonal anti-TEF-1, Rabbit polyclonal anti-MyoD, Rabbit polyclonal anti-MEF2, Mouse monoclonal anti-myogenin, Mouse monoclonal anti-α-tubulin, Mouse monoclonal anti-β-actin, Mouse monoclonal anti-GAPDH, Rabbit polyclonal anti-TEAD2, Rabbit polyclonal anti-TEAD3, and Rabbit polyclonal anti-TEAD4 antibodies used for Western blot analysis
- Positive controls: Not explicitly mentioned.
- Negative controls: Not explicitly mentioned.
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