Quantitative analysis of ferrochelatase mRNA in blood cells of erythropoietic protoporphyria patients.
Ferrochelatase (FC; heme synthetase, EC 4.99.1.1.) catalyses the synthesis of heme from protoporphyrin IX, the final step in the heme synthetic pathway. The hereditary deficiency of this enzyme gives rise to erythropoietic protoporphyria (EPP). We developed a rapid, non-radioactive means of measuring human FC mRNA levels in the EPP patients. It is based on the reverse transcriptase-polymerase chain reaction (RT-PCR) performed on the RNA obtained from peripheral blood. The amplified DNA was detected by agarose gel electrophoresis with ethidium bromide staining and the fluorescent intensity was measured by scanning densitometry applied directly to Polaroid 665 negative film. The relative expression level of FC mRNA, compared with that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, was estimated at several points in the exponential phase of PCR cycles or at a point in the exponential phase of PCR performed on serially diluted the cDNA samples. The estimate of the FC mRNA by this method correlated well with the level of the FC mRNA measured by Northern blotting in the EB virus-transformed lymphocytes of the same patients. The level of the FC mRNA appeared to vary among the patients in whom a decreased level of enzymatic activity was indicated.
Takamiyagi
A
A
Department of Dermatology, University of the Ryukyus Faculty of Medicine, Okinawa, Japan.
Nakashima
Y
Y
Irifune
H
H
Uezato
H
H
Nonaka
S
S
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D001773:Blood Cells / Q000378:metabolism*; D015152:Blotting, Northern; D002478:Cells, Cultured; D005260:Female; D005294:Ferrochelatase / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008297:Male; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017121:Porphyria, Hepatoerythropoietic / Q000097:blood* / Q000235:genetics; D012333:RNA, Messenger / Q000097:blood*
D015335:Molecular Probes; D012333:RNA, Messenger; D005294:Ferrochelatase