Quantitation

We have evaluated the quantitative potential of microarray hybridisation for both FGA (Wu et al. 2001; Rhee et al. 2004; Tiquia et al. 2004) and CGA formats (Wu et al. 2004). Linear quantitative relationships are most often observed between signal intensity and target DNA concentration over concentration ranges from 1-1,000 ng for both pure cultures and mixed target DNA populations. These results suggest that DNA microarrays may potentially be used for quantitative analysis of environmental samples. However, the difficult challenge in quantifying microbial populations in natural environments is that the community composition of the environmental samples is largely unknown. It is assumed that hybridisation signal intensity is directly related to population abundance of the target organism. However, non-specific hybridisation due to unknown diverse members in a sample may contribute to signals.

One way to assess quantitative accuracy of microarray hybridisations is the use of real-time PCR with sequence-specific primers. While it is not possible to verify an entire large microarray data set because these techniques are labour and time intensive, verification of microarray findings for several key genes is achievable and is common practice in microbial

Genes

Fig. 9.1. Correspondence of microarray-based hybridisation signals with quantitative PCR for genes associated with naphthalene degradation (Rhee et al. 2004). Quantitative PCR may be used for independent verification of key results associated with microarray studies

Genes

Fig. 9.1. Correspondence of microarray-based hybridisation signals with quantitative PCR for genes associated with naphthalene degradation (Rhee et al. 2004). Quantitative PCR may be used for independent verification of key results associated with microarray studies gene expression studies. Rhee et al. (2004) used such an approach to verify microarray hybridisation results associated with naphthalene degradation (Fig. 9.1). Overall, significant correlations between the microarray hybridisation signals and the gene copy number determined by real-time PCR were obtained with r2 = 0.74 for all genes tested, and r2 = 0.96 for the genes with SNR > 3. Such results showing consistency between microarray hybridisation data and real-time PCR data are encouraging for the use of microarray data in quantitative interpretations.

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