We found that activities of RNases are different in normal and tumor tissues. Determination of RNases activities are important not only for the extraction of full-length RNA but also for the understanding of the biological roles of RNases.
Conventional methods involve the extractions of RNases from tissues by which the spatial distribution of enzymatic activities cannot be obtained. An in situ mass spectrometric imaging is established to visualize the activities of RNases in different regions of the tissue. Normal and cancerous tissues are sliced at 10 μm with a freezing microtome. Then either a piece of synthesized marker or endogenous small fragments of RNA can be used as references. Tissue slices are uniformly sprayed and covered with a solution of DHB (2, 4-dihydroxybenzoic Acid). A Nd3+:YAG pulsed laser beam (355 nm) scans across the tissue slice with a pixel size of 20 μm. DHB is used as a matrix material that can absorb laser energy, facilitate soft ionization and protect samples from direct laser ablation. RNases present in tissues cause the degradation of intact references into fragments. Detected intensities of the fragments of references are related with the activities of RNases.
Figure 6 shows the mass spectrometric imaging of detected fragments in normal and cancerous tissue slices. It is shown there are heterogenous distribution of detected fragments GGA, GGG and CAG in tissue slices of both BALB/c 4T1 and BALB/c mice models.
Figure 6. Mass spectrometric images of three fragments in different tissue slices.
In this project, we developed a new method for the visualization of activities of RNases based on the detection of small degraded fragments. Detected fragments of RNA may result from not only in vivo RNases but also in vitro degradation.
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