Competitive RT-PCR for measuring metalloproteinase gene expression in human mesangial cells exposed to a hyperglycemic environment.
Competitive reverse transcription (RT)-PCR is now considered a reliable technique for determining the relative or absolute copy number of target mRNAs in tissue or cells. The method relies on a synthetic RNA competitor molecule with identical sequences to the PCR primers that is added to the RT step allowing reproducible quantitation between samples. In this report, we demonstrate a strategy for developing competitive RT-PCR assays using a multi-template RNA competitor molecule that is synthesized using solely PCR-based techniques. The approach we describe is both cost- and time-effective and importantly, the approach saves RNA because it allows the simultaneous quantitation of three target mRNAs from as little as one microgram of total RNA. The procedure for preparing the RNA competitor molecule, the RT-PCR protocol and the use of the competitor molecule in our studies of diabetic nephropathy are described in detail.
Death
A K
AK
Department of Medicine (Endocrinology), University of Sydney, NSW, Australia. alison@earth.endocrin.usyd.edu.au
Yue
D K
DK
Turtle
J R
JR
D000199:Actins / Q000235:genetics; D001667:Binding, Competitive; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics; D004622:Embryo, Mammalian; D015870:Gene Expression; D005920:Glomerular Mesangium / Q000187:drug effects / Q000201:enzymology*; D005947:Glucose / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases / Q000235:genetics*; D012313:RNA; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics
D000199:Actins; C034477:RNA primers; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D012313:RNA; D017364:Collagenases; D008666:Metalloendopeptidases; D020780:Matrix Metalloproteinase 9; D005947:Glucose