Construction and application of a multispecific competitor to quantify mRNA of matrix metalloproteinases and their tissue inhibitors in small human biopsies.
J Biochem Biophys Methods
; 40(3): 81-99, 1999 Aug 12.
Article
in En
| MEDLINE
| ID: mdl-10487163
ABSTRACT
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.
Search on Google
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA, Messenger
/
Tissue Inhibitor of Metalloproteinases
/
Reverse Transcriptase Polymerase Chain Reaction
/
Matrix Metalloproteinases
Limits:
Female
/
Humans
Language:
En
Journal:
J Biochem Biophys Methods
Year:
1999
Type:
Article
Affiliation country:
Australia