I am investigating the therapeutic role of miRNAs in fibrosis (in vivo). Being aware of the importance of the balance/imbalance between Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases, what would be the best way to show the change in Mmp vs. Timp? mRNA, protein, enzymatic activity? The samples I have are: murine liver total RNA, serum, frozen samples for protein extraction, and paraffin wax embedded livers. Thank you for advice.
I think that using q-RT-PCR + WB of liver tissue would be more than enough. You also can perform enzymatic tests and IHQ ... justo to show MMP distribution on tissue.
If you itend to show fibrosis reduction, besides showing results from morphometry (quantification of fibrosis areas), you could measure some fibrosis markers in the serum: Hyaluronic Acid, Laminin, PIIIP, AST:ALT ratio....Good luck!
The best assay is in-situ zymography, for that you would need fresh tissue samples or frozen in OCT to maintain enzymatic activity. check out my 2004 JCI paper ad let me know if you need other info.
Ciao
depends on what is the routine technique used in your lab. WB analysis of the frozen samples to evaluating the levels of the specific MMP changed in fibrosis, I don't know may be MMP-9 or MMP-3, in comparison to the levels of TIMP-3 would be a start.
www.biology-online.org/articles/metabolic.../zymography.html
Biology online suggests that geletine zymography is the best technique to study the MMP/TIMP relationship using snadwich ELISA techniques
. I have copied the method from the website below.
Zymography
Zymography is a simple, sensitive, quantifiable, and functional approach for the analysis of proteolytic activity in cell and tissue extracts, which was introduced more that 20 years ago (Heussen and Dowdle 1980). It is widely used to study extracellular matrix (ECM)-degrading enzymes, in particular the MMPs. MMPs are zinc-dependent endopeptidases capable of degrading ECMs, including the basement membrane. The MMP family consists of at least 26 members and has been classified into subgroups on the basis of substrate preference and molecular structure. These are interstitial collagenases, gelatinases, stromelysins, and matrilysins, although all enzymes have overlapping substrate specificity. A recently discovered MMP family consists of membrane-type MMPs that are characterized by a transmembrane domain or a GPI-anchor (Visse and Nagase 2003).
The standard method for zymography is based on the use of SDS-polyacrylamide gels co-polymerized with a protein substrate, in particular gelatin, casein, or fibrin. Proteases that have the ability to renature after removal of SDS and to exert proteolytic activity on a co-polymerized substrate can be analyzed with this method. MMP-2 (gelatinase A, 72 kD) and MMP-9 (gelatinase B, 92 kD) can be detected on gelatin zymograms and MMP-7 on casein gels. Coomassie Blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Figure 2 shows a zymogram of a homogenate of a tumor of colon cancer cells in mouse liver containing four bands responsible for gelatin breakdown. Based on the molecular weights, these bands reflect inactive and active MMP-2 and inactive and active MMP-9 (Ackema, unpublished results). Polyacrylamide gel co-polymerization with plasminogen and gelatin allows detection of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) as plasmin generated by uPA and/or tPA degrades gelatin (Figure 3 ; Leber and Balkwill 1997). Casein zymography has been used as an alternative assay for fibrinolytic enzymes because of the ability of plasmin to degrade casein. Kim et al. (1998) introduced fibrin zymography to detect fibrinolytic enzymes by incorporating fibrinogen and thrombin into SDS polyacrylamide gels. Although gelatin and casein are satisfactory substrates for plasmin, all fibrinolytic enzymes are not able to cleave these substrates as plasmin is. Apparently, fibrin is an in vivo substrate for plasmin and plasmin-like enzymes that can be demonstrated reliably with fibrin zymography.
Zymography offers advantages over other methods, such as ELISA. Expensive materials are not required (e.g., antibodies) and proteases with different molecular weights showing activity towards the same substrate can be detected and quantified on a single gel. For example, MMPs are released from cells in a proteolytically inactive proform (zymogen) which is approximately 10 kD larger than the activated form. Because the proform becomes activated during the process of denaturation and renaturation after gel electrophoresis, the active form and the originally inactive forms degrade gelatin, and both forms can therefore be detected on zymograms. In addition, MMPs in solution are often associated with endogenous tissue inhibitors of metalloproteases (TIMPs). During electrophoresis the inhibitors dissociate from the MMP and do not interfere with detection of the enzymatic activity. On the other hand, sandwich ELISA can discriminate between MMP/TIMP complexes and free MMPs, resulting in determination of a potential active fraction (Zucker et al. 1992; Ratnikov et al. 2002; Catterall and Cawston 2003).
It can be concluded that zymography enables the detection of protease activity in cell or tissue homogenates using gelatin, casein, or fibrin as substrates. On the basis of molecular weight markers, the molecular weight of the proteolytic band can be determined, and by comparison with recombinant proteins and the use of specific protease inhibitors the type of protease can be established. However, information on the localization of the proteolytic activity in cells or tissues cannot be obtained on the basis of zymography.
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