Real-time RT-PCR discriminating mRNA encoding osteocalcin from unspecific targets.
Osteocalcin is a noncollagenous protein produced by osteoblasts and odontoblasts. It is used as a marker for bone formation in regenerative medical approaches. In addition, serum levels in humans are used to indicate bone turnover. Expression is usually determined by real-time reverse transcription PCR. Analysis of sequence data revealed that the frequently used primers for the determination of osteocalcin expression also detect the expression of messenger RNA (mRNA) encoding polyamine-modulated factor 1 (PMF1). In the present study we developed a method to determine the real amount of mRNA encoding osteocalcin. Bone-derived cells were treated with osteogenic differentiation medium and expression of osteocalcin was determined to test the method. It was found that the classic method that does not correct for PMF1 expression leads to overestimations as high as 70%.
Burkhardt
Jan-Karl
JK
Institute of Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103, Leipzig, Germany.
Halama
Dirk
D
Frerich
Bernhard
B
Gaunitz
Frank
F
D001483:Base Sequence; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics
C120655:PMF1 protein, human; D012333:RNA, Messenger; D014157:Transcription Factors; D015675:Osteocalcin