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.
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/micrograms 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.
D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004277:DNA, Single-Stranded; D005069:Evaluation Studies as Topic; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics; D018168:Receptors, Retinoic Acid; D012867:Skin / Q000737:chemistry / Q000166:cytology; D014212:Tretinoin / Q000494:pharmacology*
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D012313:RNA