Construction and application of a multispecific competitor to quantify mRNA of matrix metalloproteinases and their tissue inhibitors in small human biopsies.
Accurate quantitation of mRNA levels of a number of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in very small samples such as human biopsy material has not been generally possible. This paper describes the development, validation and application of a quantitative RT-PCR (Q-RT-PCR) assay that allows the detection and quantitation of mRNAs encoding genes of three MMPs (MMP-1, MMP-2, MMP-3), three TIMPs (TIMP-1, TIMP-2, TIMP-3) and GAPDH simultaneously from small amounts of RNA (< 4 microg). A multispecific competitor which shares the same primer-binding sequences as the cellular mRNA of all seven genes, but yields different sized PCR products, was constructed by adding primers specific for the MMPs and TIMPs to a core molecule (mutated GAPDH) by sequential PCR and cloning, and its multispecificity was experimentally validated. Application of the technique to measurement of transcriptional levels of MMPs and TIMPs in cultured human endometrial stromal cells provided support to the hypothesis that progesterone withdrawal alters the ratio of MMPs to TIMPs in favor of MMPs. This Q-RT-PCR method is a relatively simple, highly specific and nonradioactive procedure and is widely applicable.
Nie
G Y
GY
Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia. guiying.nie.@med.monash.edu.au
Wang
J
J
Li
Y
Y
Salamonsen
L A
LA
D001483:Base Sequence; D001667:Binding, Competitive; D001706:Biopsy; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004717:Endometrium / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D020782:Matrix Metalloproteinases / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D020782:Matrix Metalloproteinases