Use of the polymerase chain reaction in quantification of interleukin 8 mRNA in minute epidermal samples.
Quantitative studies of cytokine gene expression in vivo are necessary in order to properly describe the cytokine network and to elucidate its role in skin inflammation. Ideally, one should be able to follow cytokine gene expression in epidermal, dermal, and blood compartments. However, such studies are limited by small amounts of available material. Here we report a polymerase chain reaction (PCR) cDNA amplification protocol useful for quantification of specific mRNAs in small skin samples. We found that analysis of dilution series of each sample permitted establishment of quantitative PCR amplification conditions using only picogram to nanogram amounts of total RNA. Cytokine mRNA amounts could then be measured relative to an internal standard species, co-reverse transcribed, and co-amplified with the cytokine species as a measure of cDNA input. Large numbers of samples can be screened rapidly with initial short dilution series identifying cytokine-positive samples and the correct dilution range for each, followed by closer analysis in this range. Epidermal samples obtained through curettage of a small skin area, 2-mm dermal biopsies from the scraped sites, and a few blood drops from the biopsy sites all yielded sufficient RNA for analysis by this protocol. Any mRNA of known sequence can be studied. We analyzed interleukin 8 mRNA levels in more than a hundred epidermal samples from patients and normal test persons and found a variation over several orders of magnitude that seemed to follow the degree of inflammation of the skin.
Paludan
K
K
Department of Molecular Biology, Marselisborg Hospital, University of Arhus, Denmark.
Thestrup-Pedersen
K
K
D001483:Base Sequence; D004247:DNA / Q000032:analysis; D003877:Dermatitis, Contact / Q000378:metabolism; D004817:Epidermis / Q000737:chemistry*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011565:Psoriasis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012867:Skin / Q000737:chemistry
D016209:Interleukin-8; D012333:RNA, Messenger; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases