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Nucleic Acids Research, 1992, Vol. 20, No. 23 6215-6220
© 1992

METHODS

Quantitation of human cellular retinoic acid-binding protein II (CRABP-II) RNA from cultured human skin fibroblast cells and human skin biopsies treated with retinoic acid

Lubing Zhou, Gail Otulakowski, Jesse Pang, Donald G. Munroe, Robert J. Capetola1 and Cathy Lau

The R.W.Johnson Pharmaceutical Research Institute 19 Green Belt Drive, Don Mills, Ontario M3C 1L9, Canada 1The R.W.Johnson Pharmaceutical Research Institute 920 US Route #202, Raritan, NJ 08869-0602, USA

Received September 28, 1992. Accepted November 2, 1992.

A polymerase chain reaction (PCR) method has been validated for the quantitation of retinoic acid (RA) induction of cellular retinoic acid-binding protein II (CRABP-II) RNA from cultured human skin fibroblasts and human skin biopsies. The method utilizes reverse transcription and PCR (RT-PCR) to compare cellular CRABP-II RNA with a known amount of added internal standard RNA generated from a modified CRABP-II cDNA containing a 42 bp deletion. Thus, after RT-PCR of cellular and standard CRABP-II RNA in the same tube, the resulting DNA bands could be distinguished by size on ethidium bromide-stained, nondenaturing polyacrylamide gel. Serial dilutions of cellular RNA were co-amplified with a fixed amount of internal standard CRABP-II RNA, and the ratio of intensities of the two DNA bands was determined by computerized image analysis of the gel photograph. A linear relationship was found between the logs of this ratio and the input RNA. Absolute quantitation of cellular CRABP-II RNA was determined from the ‘equivalence point’, the dilution at which band intensities from cellular and standard RNAs were identical. Using this quantitative assay, the amount of CRABP-II RNA in cultured fibroblasts was 24 attomoles per microgram total RNA. A 4.2-fold increase in CRABP-II RNA was seen following 24 hours treatment with 10–6 M RA. CRABP-II RNA content in skin biopsies taken from 3 human subjects ranged from 16 to 25 attomole/µg RNA. Topical treatment with 0.1% RA cream resulted in induction ranging from 3.9- to 12-fold over vehicle treatment. The method described here offers a rapid, sensitive and quantitative assay of specific RNAs, and should be especially useful for the measurement of RNA levels from small solid-tissue biopsies.


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