.png){kind=icon}
1.1 What is meant by the term ischaemia and what are the two ways in which cells suffer degradation?
The term ischaemia is used to define the interval between removal of nutrient and blood supply from cells/tissues and the placing of these in a preservative or fixative. During the ischaemic phase the cytology and molecular preservation of samples may be compromised by autolysis and/or putrefaction.
1.2 What are the main routes for the preparation of tissue for morphological analysis and blood cells for nucleic acid analysis following extraction?
The main route for the preparation of tissue for morphological analysis involves formalin fixation and paraffin wax embedding. This conserves the majority of cellular constituents and preserves them in their location allowing subsequent microscopic viewing. In contrast blood cells destined for nucleic acid analysis after extraction are collected in an appropriate anticoagulant after which a protocol is used to extract the nucleic acids. In this situation no cytological preservation of the sample is attempted.
1.3 What factors may determine the quality of protein and nucleic acid extracts?
Factors affecting the quality of protein and nucleic acid extracts are determined by handling before, during and after extraction. Prior to extraction keeping time intervals short and using appropriate stabilising measures such as anticoagulants and transport media will promote quality. Exposure to formalin compromises the quality of proteins and nucleic acids, but such samples can be used providing modified extraction methods are employed. During any extraction procedure it is important to protect constituents from further degradation. This is particularly important for RNA that is easily degraded by the ubiquitous presence of RNases. Lastly the use of appropriate storage conditions will assist in conserving extracts.
1.4 What methods are available to assess the quality of DNA in intact cellular and extract preparations?
For intact cellular preparations simultaneous in situ hybridisation is often undertaken using a centromeric or peri-centomeric ‘control’ probe present on the same chromosome as the gene of interest. For the assessment of the quality of DNA in extracts a variety of methods are available. These include UV spectrophotometric, gel electrophoretic, fluorescent methods and the use of PCR. An important distinction between assessment of DNA in intact cellular and extract preparations is that with the latter quantitation of the amount of recovered nucleic acid is also provided.