Quantitation of matrix Gla protein mRNA by competitive polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control.
Matrix Gla (gamma-carboxyglutamic acid) protein (MGP) is a vitamin-K-dependent extracellular matrix protein. A method was developed to quantitate MGP mRNA based on competitive polymerase chain reaction following reverse transcription (competitive RT-PCR). The MGP cDNA was coamplified with a mutant MGP cDNA (competitor). The ratio of MGP to competitor after the PCR reaction was compared to standards to determine the amount of MGP mRNA in RT samples. MGP mRNA in as little as 3.125 ng total RNA was accurately quantitated and was far more sensitive than RNA hybridization methods. To control for variations due to sample preparation, a second competitive RT-PCR was developed to measure the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from the same sample as an internal control. Thus, the amount of MGP is normalized to the amount of the housekeeping gene GAPDH. The accuracy, sensitivity and ease of this new method enables rapid mRNA quantitation without blotting, hybridization or autoradiography. The method is particularly advantageous for MGP mRNA measurement from a small amount of sample. Using this assay, we established that MGP mRNA increases approx. fivefold with co-treatment of retinoic and ascorbic acids.
Zhao
J
J
Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
Araki
N
N
Nishimoto
S K
SK
D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis* / Q000235:genetics; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D000097568:Matrix Gla Protein
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012333:RNA, Messenger; D000097568:Matrix Gla Protein