EXTRACTION AND QUANTITATION OF CYTOKINE MESSENGER-RNA FROM HUMAN EPIDERMAL BLISTER ROOFS

被引:7
作者
ENK, CD [1 ]
KATZ, SI [1 ]
机构
[1] NCI,DERMATOL BRANCH,BETHESDA,MD 20892
关键词
HUMAN EPIDERMIS; POLYMERASE CHAIN REACTION; CYTOKINES;
D O I
10.1007/BF00370722
中图分类号
R75 [皮肤病学与性病学];
学科分类号
100206 ;
摘要
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-mu l 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.
引用
收藏
页码:72 / 77
页数:6
相关论文
共 27 条
[1]  
CHELLY J, 1989, P NATL ACAD SCI USA, V86, P2517
[2]   ANTIGEN-PRESENTING OKM5+ MELANOPHAGES APPEAR IN HUMAN-EPIDERMIS AFTER ULTRAVIOLET-RADIATION [J].
COOPER, KD ;
NEISES, GR ;
KATZ, SI .
JOURNAL OF INVESTIGATIVE DERMATOLOGY, 1986, 86 (04) :363-370
[3]  
CROSSMAN RM, 1989, P NATL ACAD SCI USA, V86, P6367
[4]   CYTOKINE GENE-EXPRESSION - ANALYSIS USING NORTHERN BLOTTING, POLYMERASE CHAIN-REACTION AND INSITU HYBRIDIZATION [J].
DALLMAN, MJ ;
MONTGOMERY, RA ;
LARSEN, CP ;
WANDERS, A ;
WELLS, AF .
IMMUNOLOGICAL REVIEWS, 1991, 119 :163-179
[5]   QUANTITATIVE MEASUREMENT OF MESSENGER-RNAS BY POLYMERASE CHAIN-REACTION [J].
DELIDOW, BC ;
PELUSO, JJ ;
WHITE, BA .
GENE ANALYSIS TECHNIQUES, 1989, 6 (06) :120-124
[6]   LOCALIZATION AND CHARACTERIZATION OF THE INTERLEUKIN-1 IMMUNOREACTIVE POOL (IL-1 ALPHA AND BETA FORMS) IN NORMAL HUMAN-EPIDERMIS [J].
DIDIERJEAN, L ;
SALOMON, D ;
MEROT, Y ;
SIEGENTHALER, G ;
SHAW, A ;
DAYER, JM ;
SAURAT, JH .
JOURNAL OF INVESTIGATIVE DERMATOLOGY, 1989, 92 (06) :809-816
[7]  
EHRLICH GD, 1990, PCR PROTOCOLS GUIDE, P325
[8]   OVEREXPRESSION OF TRANSFORMING GROWTH FACTOR-ALPHA IN PSORIATIC EPIDERMIS [J].
ELDER, JT ;
FISHER, GJ ;
LINDQUIST, PB ;
BENNETT, GL ;
PITTELKOW, MR ;
COFFEY, RJ ;
ELLINGSWORTH, L ;
DERYNCK, R ;
VOORHEES, JJ .
SCIENCE, 1989, 243 (4892) :811-814
[9]   EARLY MOLECULAR EVENTS IN THE INDUCTION-PHASE OF CONTACT SENSITIVITY [J].
ENK, AH ;
KATZ, SI .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (04) :1398-1402
[10]  
HAUSER C, 1986, J IMMUNOL, V136, P3317