Frozen tissue, analysis

Mass Spectrometry Analysis for Protein MALDI-MSI (For Frozen Tissue Analysis Exclusively)... 

When fresh or frozen tissue is used for proteomic analyses, the results cannot be related directly to the clinical course of diseases in a timely manner. Instead, researchers frequently reduce the number of interesting proteins to a manageable number and then attempt to use immunohistochemistry to understand the implications of proteomic changes in archival formalin-fixed, paraffin-embedded (FFPE) tissue for which the clinical course has been established.3 Unfortunately, immunohistochemistry is a semiquantitative pro-teomic method, and the choice of interesting proteins must occur without advance knowledge of the clinical course of the disease or the response to therapy. If routinely fixed and embedded archival tissues could be used for standard proteomic methods such as 2-D gel electrophoresis and mass spectrometry (MS), these powerful techniques could be used to both qualitatively and quantitatively analyze large numbers of tissues for which the clinical course has been established. However, analysis of archival FFPE tissues by... 

A study of by Palmer-Toy et al.,12 summarized in Table 19.1, provides further empirical evidence of the utility of techniques coupling heating with efficient protein extraction for the proteomic analysis of FFPE tissue. A specimen from a patient with chronic stenosing external otitis was divided in half and preserved as fresh-frozen tissue or FFPE. Ten micromolar sections of the FFPE tissue were vortexed in heptane to deparaffinize the tissue and were then co-extracted with methanol. The methanol layer was evaporated, and the protein residue was resuspended in 2% SDS/lOOmM ammonium bicarbon-ate/20mM dithiothreitol (DTT), pH 8.5 and heated at 70°C for lh. After tryptic digestion, 123 total confident proteins were identified in the FFPE tissue, compared to 94 proteins identified from the fresh-frozen tissue. Hwang et al. also reported up to a fivefold increase in protein extraction efficiency for samples extracted in a Tris-HCl/2% SDS/1% Triton X-100/1% deoxycholate solution at 94°C for 30 min versus samples extracted in 100 mM ammonium bicarbonate/30% acetonitrile at the same temperature.14... 

For imaging MS analysis, animals were sacrificed, and the extirpated brains were immediately frozen in powdered dry ice and stored at -80°C until needed. Briefly, fresh-frozen tissues were sliced into 5-pm-thin sections and mounted... 

This multiprobe RPA offers the advantages of its sensitivity and capacity to simultaneously quantitate several mRNA species in a single sample of total RNA. This allows comparative analysis of different mRNA species within samples, moreover, by incorporating probes for housekeeping gene transcripts, the levels of individual mRNA species can be compared between samples. Furthermore, the assay is highly specific and quantitative owing to the RNase sensitivity of mismatched base pairs and the use of solution-phase hybridization driven toward completion by excess probe. Finally, the multiprobe RPA can be performed on total RNA preparations derived by standard methods from either frozen tissues or cultured cells. 

Karsten SL, Van Deerlin VM, Sabatti C et al. An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis. Nucleic Acids Res 2002 30 E4. 

Usually no ADP can be seen in NMR spectra, although there may be about 0.5 mM ADP according to chemical analysis of rapidly frozen tissues. It has been concluded that most ADP is bound to proteins. 

Shapiro C, Crosby K, Lewis M et al. Optimization and Validation of Activation-State Specific Antibodies for the Immunohistochemical Analysis of Fresh Frozen Tissues and Cells. Proc Am Assoc Cancer Res. 2006. 

The sample preparation procedures for the direct analysis of small molecules in tissue have been described by several papers [120-124], Tissues (brain, heart, lung, kidney, liver, etc.), were immediately frozen and stored at -80 °C after harvest. The frozen tissues were subsequently cut into serial 10-20 pm thick section which was typically prepared by cryosectioning on a microtome at a temperature of -20 °C. The adjacent sections were gently mounted onto a conductive surface, MALDI imaging target plate or glass slides. These plates were desiccated under low vacuum for a short period of time until dry, then robotically or manually coated with the... 

Free radicals can be detected by direct ESR (room temperature and low temperature) and spin trapping. Room-temperature ESR measurements in heart tissues are not feasible using conventional ESR because of the high electric constant of water, which is a major component of heart tissues. Therefore, one is forced to use low-temperature ESR measurements for heart tissues. An apparent drawback with low-temperature ESR, especially as it relates to detecting free radicals, is that the frozen tissue must be mechanically processed prior to ESR analysis. This has caused a considerable amount of confusion and erroneous conclusions in the myocardial free radical literature, as will be discussed below. 

As noted earlier, plasma from blood samples must be promptly stabilized and, if necessary, the acidified samples may be stored frozen at — 65°C. Because of the existence of oxyhemoglobin in whole blood or red cell suspensions, some consideration must be given to inactivate oxyhemoglobin or use an assay for total ascorbic acid content. With respect to tissue analysis, some discretion must be considered as to the degree of blood contamination. 

Lysates of frozen tissue containing 900 pg of protein were immunoprecipitated with specific monoclonal antibody. The resultant immunocomplexes were resolved in 12.5% polyacrylamide slab gels containing SDS Western blotting was performed. After electrophoretic transfer of protein from the gel onto the nitrocellulose filter, protein was detected by the use of polyclonal antibody and 125I-labeled protein A. Quantitative differences in the levels of protein are determined by densitometric analysis. 

Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. RT-PCR performed on fresh-frozen tissue provides high-quality amplification and reliable results. However, when FFPE tissue is used for RT-PCR analysis, the results vary and depend on the level of RNA degradation and length of PCR amplification. To attain a more stable RT-PCR amplification from FFPE tissues, it is typical to choose a target that is less than 150 to 200 nt long. 

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