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 metalloproteinas es (MMPs) and their tissue inhibitors (TIMPs) in very small samples such as human biopsy material has not been generally possible. This paper describe s the development, validation and application of a quantitative RT-PCR (Q-R T-PCR) assay that allows the detection and quantitation of mRNAs encoding g enes 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 mu g). A multisp ecific competitor which shares the same primer-binding sequences as the cel lular 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 mo lecule (mutated GAPDH) by sequential PCR and cloning, and its multispecific ity was experimentally validated. Application of thr technique to measureme nt of transcriptional levels of MMPs and TIMPs in cultured human endometria l stromal cells provided support to the hypothesis that progesterone withdr awal alters the ratio of MMPs to TIMPs in favor of MMPs. This Q-RT-PCR meth od is a relatively simple, highly specific and nonradioactive procedure and is widely applicable. (C) 1999 Elsevier Science B.V. All rights reserved.