Absence of calbindin-D28 expression in nonclassical 1,25-dihydroxyvitamin D targets: analysis by polymerase chain reaction.
CaBP-D28 mRNA expression in rat heart, testis, and lung was assessed by polymerase chain reaction (PCR). The animal model used was the hyperinduced vitamin D-treated rat (100 ng 1,25-dihydroxyvitamin D subcutaneously, daily for 7 days). For the PCR studies, two pairs of 20 mer oligonucleotide primers (designated 1-4 according to their position on the coding strand, but with primers 3 and 4 in reverse orientation) derived from the rat CaBP-D28 cDNA sequence were tested in various combinations. Optimal conditions were established using a 1:100 dilution of cDNA from normal rat kidney. Bands of the predicted sizes of 869 (1, 3), 994 (1, 4), 725 (2, 3), and 850 (2, 4) nucleotide base pairs resulted, but with varying intensities: 2,4 approximately 1,3 > 1,4 > 2,3. Repeat PCR (recycling after 1:100 dilution and readdition of reagents and primers with at least one different primer) provided strong additional amplification, particularly with the 1,4/2,4 combination. Under these conditions, mixing experiments showed that CaBP-D28 transcripts were detectable at 10(-7)- to 10(-9)-fold lower levels of expression than in D+ kidney. When RNA was isolated and cDNA generated from test tissues from 4 individual vitamin D-stimulated (D+) and vitamin D-deficient (D-) rats, repeat PCR (1,4/2,4 primer combination) provided no evidence of significant CaBP-D28 mRNA expression in the nonclassic target tissues, in contrast to strong bands in both the D- kidney (undiluted) and D+ kidney (1:100 dilution) preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Walters
M R
MR
Central Research Unit, F. Hoffmann-La Roche, Ltd., Basel, Switzerland.
Hunziker
W
W
D000818:Animals; D001483:Base Sequence; D064026:Calbindins; D002117:Calcitriol / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry; D007668:Kidney / Q000378:metabolism; D008168:Lung / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis* / Q000235:genetics; D013737:Testis / Q000378:metabolism*; D014808:Vitamin D Deficiency / Q000378:metabolism
D064026:Calbindins; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D004247:DNA; D002117:Calcitriol