Extraction and quantitation of cytokine mRNA from human epidermal blister roofs.
The demonstration of cytokine mRNA expression in epidermal cells by the polymerase chain reaction technique preceded by reverse transcription (RT-PCR) requires linear test conditions (i.e. that the product obtained after amplification reflects the relative amounts of starting material) and high reproducibility, specificity, and sensitivity. By combining well-defined techniques for mRNA extraction and concentration measurement with a sensitive and well-calibrated RT-PCR technique, we demonstrated the presence of IL-1 alpha, IL-1 beta, IL-6, and TNF alpha in epidermal cells obtained from suction blister roofs from normal volunteers. Messenger RNA was extracted with superparamagnetic oligo(dT)25 Dynabeads, and the amount of mRNA was measured by spectrophotometry using a Beckman 5-microliters Ultra-Microcell prior to RT-PCR. Linear PCR conditions were obtained by carefully titrating the amounts of mRNA and the number of cycles. Reproducibility was estimated at different steps of the procedure, and the specificity of the enhanced cDNA products was verified by liquid hybridization with end-labelled probes. We suggest that this combination of techniques might prove useful for the simultaneous assessment of the expression of various cytokines from small samples of fresh human epidermal cells.
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Dermatology Branch, National Institute of Health, National Cancer Institute, Bethesda, MD 20892, USA.
Katz
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D001768:Blister / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis*; D000078404:Epidermal Cells; D004817:Epidermis / Q000737:chemistry*; D006801:Humans; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D013053:Spectrophotometry
D016207:Cytokines; D012333:RNA, Messenger