Total RNA from KCs or frozen skin was isolated using RNeasy mini kits with on-column DNase digestion (Qiagen, Valencia, CA). Total RNA was reverse transcribed using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit. cDNA equivalent to 5–40 ng of total RNA was used for QRT- PCR using pre-validated TaqMan gene expression assays (Applied Biosystems, Foster City, CA) for AREG (# Hs00155832), BTC (# Hs00156140), EREG (# Hs00914313, EPGN (# Hs02385425), HB-EGF (# Hs00181813, TGF-α (# Hs00608187) and ribosomal protein large P0 (RPLP0 or 36B4, # Hs99999902) (Laborda, 1991 (link); Minner and Poumay, 2008 ). Data are expressed as fold-change relative to 36B4 multiplied by 103 (fold-change versus 36B4 = 2 –(CT target -CT 36B4)).
Protocol detail
Quantifying Epidermal Growth Factors
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Areg
Assays
Cdna
Digestion
Dnase
Ereg
Frozen
Gene expression
Hb egf
Reverse transcription
Ribosomal protein p0
Rplp0
Skin
Tgf α
Corresponding Organization :
Other organizations : University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Total RNA from KCs or frozen skin
- HB-EGF
- TGF-α
- Ribosomal protein large P0 (RPLP0 or 36B4)
- Positive control: Not specified
- Negative control: Not specified
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