Mutation analysis

Fig. 9.1 The principle and applications oflaser microdissection technology. In laser microdissection laser beam is used to dissect small cell populations from paraffin or frozen sections. Cells can then be used to ordinary DNA, RNA and protein analysis.

Tab. 9.1 Minimal criteria for an array analysis in cardiovascular field

- Findings have to be confirmed by other, independent methods such as RT-PCR, Northern blot, immunohistochemistry or in situ hybridisation

- Findings must be reproducible i.e. confirmed with repeated hybridisations (at least 3 times) using multiple sources of RNA

- statistical methods must be used to interpret the data

GenBank. Comparative databases in www should include the possibility to 1) search for the data by name of the gene, 2) find the expression value corrected to a uniform format, 3) have an access to brief annotations of specific genes and links to known biochemical pathways, including interactions at the transcriptional level, 4) download data for further analyses and 5) generate reports [3].

Atherosclerotic plaques can usually be divided to macrophage-rich shoulder areas, fibrous cap and atheromatous core. By manual dissection it is not possible to adequately separate these parts, and one has to be satisfied with heterogenous samples containing various parts of the lesion, media and adventitia. Recently, a novel method named "laser microdissection" has been developed to dissect very small cell populations or even single cells. Thin (<5 ^m) frozen or paraffin sections are cut to special plastic slides, stained and dissected by laser beam under microscopic control [28]. RNA, DNA and proteins can be extracted from microdis-sected cells and used for PCR and DNA-array analyses (Fig. 9.1). Because the amounts of RNA that can be extracted are very low, T7-RNA-polymerase-based amplification is needed before any array analyses can be performed [29].

Tab. 9.1 states the minimal criteria for an array analysis in cardiovascular field. Analyses have to be repeated at least three times and statistical methods need to be used to interpret the data. Findings must be confirmed by complementary methods such as RT-PCR or immunohistochemistry since DNA arrays may still produce false signals due to sequence homologies and repetitive elements present in many genes.

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