Total RNAs were extracted by trizol (Invitrogen) and cDNAs were synthetised using Rever Ace qPCR RT Kit (TOYOBO). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix (Roche) and the ABI ViiA7 QPCR System (Applied Biosystems). Chromatin immunoprecipitation (ChIP) was performed to investigate whether EZH2 or/and EZH2 binding to HOXA9 promoter. ChIP assays were performed as described previously (38 (link)). Final analysis was performed using qPCR and shown as fold enrichment of the HOXA9 gene promoter.
Protocol detail
Analyzing EZH2 Binding to HOXA9 Promoter
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Assays
Cdnas
Chromatin immunoprecipitation
Ezh2
Gene promoter
Hoxa9
Realtime pcr
Rnas
Sybr green
Trizol
Corresponding Organization : Tongji Hospital
Other organizations : Union Hospital, Hubei University of Chinese Medicine, University of California, Los Angeles, The Wistar Institute
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Protocol cited in 2 other protocols
Variable analysis
independent variables
- EZH2 binding to HOXA9 promoter
- Fold enrichment of the HOXA9 gene promoter
- Trizol (Invitrogen) for total RNA extraction
- Rever Ace qPCR RT Kit (TOYOBO) for cDNA synthesis
- SYBR Green Realtime PCR Master Mix (Roche) for real-time PCR
- ABI ViiA7 QPCR System (Applied Biosystems) for real-time PCR
- ChIP assays performed as described previously (38)
- Positive control: Not specified
- Negative control: Not specified
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