Total RNA extraction and cDNA synthesis were performed as previously described.30 (link) PCR reactions were performed in duplicate using the ABI 7500 Fast Real-Time PCR and TaqMan gene expression assays (Applied Biosystems). The following TaqMan gene probes were used: Ppargc1a, Ndufs1, Cycs, Atp5o, Nrf1, Tfam, Vegfa, Nos1, Nos3, Hcar2. Of the four known Ppargc1a transcripts (1–4), Ppargc1a1 (Taqman Mm00447183_m1) was studied in all gene expression analyses.38 (link) Mouse Ido2, Afmid, Kynu, Kmo, Haao, Qprt, Naprt, and Nmnat1 for SYBR Green PCR have been described elsewhere.39 (link),40 (link) Mouse Nampt SYBR primers were designed using PrimerQuest Tool (Integrated DNA Technologies). Relative expression levels were determined using the comparative threshold method.
Protocol detail
Comprehensive Gene Expression Analysis
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Assays
Cdna
Gene
Gene expression
Gene expression analyses
Mouse
Nampt
Nos1
Nos3
Ppargc1a
Primers
Real time pcr
Sybr green
Synthesis
Tfam
Corresponding Organization :
Other organizations : Beth Israel Deaconess Medical Center, Harvard University, Center for Vascular Biology Research, Massachusetts General Hospital, Broad Institute
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Protocol cited in 1 other protocol
Variable analysis
independent variables
- Ppargc1a
- Ndufs1
- Atp5o
- Vegfa
- Hcar2
- Afmid
- Naprt
- Nmnat1
- Nampt
- Gene expression levels
- Total RNA extraction and cDNA synthesis were performed as previously described.
- PCR reactions were performed in duplicate
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