Rapid and sensitive method of quantitation of bone gla protein mRNA using competitive polymerase chain reaction.
A method for sensitive quantitation of bone gla protein (BGP, osteocalcin) mRNA has been developed using competitive polymerase chain reaction after reverse transcription (competitive RT-PCR). The complementary DNA (cDNA) were transcribed from sample RNA was co-amplified in a PCR with a known amount of mutant BGP cDNA (competitor) using the identical oligonucleotide primers. The mutant cDNA with its unique restriction site allowed quantitation of sample and mutant PCR products after densitometric analysis of ethidium bromide-stained agarose gels. A linear relationship between initial sample BGP amount and the ratio of BGP to mutant BGP band intensity was obtained and used to make a standard curve to determine the initial BGP mRNA of unknown samples. These standard curves were made with known amounts of recombinant BGP cDNA. The competitive RT-PCR for BGP allows measurement of twofold differences in 1 and 2 micrograms total RNA and requires at least 10 times less sample RNA than usual Northern blotting. Moreover, heteroduplexes with one BGP strand and one mutant BGP strand formed as a result of high PCR cycles were quantifiable. This provided the advantages of rapid quantitation from ethidium bromide-stained gels without blotting, hybridization, or autoradiography. Multiple samples could be assayed for greater confidence in the results. The sensitivity, accuracy, and ease of the assay will facilitate analysis of BGP mRNA from a small amount of sample. The assay has been used to confirm the BGP mRNA changes with hormonal treatment in cultured cells and the age-related changes in whole tibia in vivo.
Araki
N
N
Department of Biochemistry, University of Tennessee, Memphis.
Robinson
F D
FD
Nishimoto
S K
SK
D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000737:chemistry*; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009154:Mutation; D015675:Osteocalcin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011916:Rats, Inbred F344; D014158:Transcription, Genetic
D012333:RNA, Messenger; D015675:Osteocalcin; D004247:DNA