Assessing the Reliability of Amplified RNA Used in Microarrays

A DUMB Table Approach

Abstract

A certain minimal amount of RNA from biological samples is necessary to perform a microarray experiment with suitable replication. In some cases, the amount of RNA available is insufficient, necessitating RNA amplification prior to target synthesis. However, there is some uncertainty about the reliability of targets that have been generated from amplified RNA, because of nonlinearity and preferential amplification. This current work develops a straightforward strategy to assess the reliability of microarray data obtained from amplified RNA. The tabular method we developed, which utilises a Down-Up-Missing-Below (DUMB) classification scheme, shows that microarrays generated with amplified RNA targets are reliable within constraints. There was an increase in false negatives because of the need for increased filtering. Furthermore, this analysis method is generic and can be broadly applied to evaluate all microarray data.

A copy of the Microsoft® Excel® spreadsheet is available upon request from Edward Bearden.

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Acknowledgements

The authors wish to thank Radhakrishnan Nagarajan, Richard Dennis and Eric Siegel from the University of Arkansas for Medical Sciences (UAMS) for their input and support. Also, appreciation is expressed to the National Science Foundation (NSF) Research Coordination Network for facilitating the interchange of ideas in microarray analysis.

This research was supported in part by funds provided to the UAMS Microarray Facility through Act 1, The Arkansas Tobacco Settlement Proceeds Act of 2000, and by NIH grant no. P20 RR-16460 from the Biomedical Research Infrastructure Network (BRIN) Program of the National Center for Research Resources.

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Correspondence to Dr Edward D. Bearden.

Appendices

Appendix A

Sequences for the primer pairs are given in table AI.

Table AI
figureTabAI

Primer sequences for the genes selected for verification by reverse transcription PCR

Appendix B: A Comparison of Dissimilar Slides

In order to see how the DUMB table responds when comparing two dissimilar arrays, we compared another pair of dye-swapped slides. However, in this case, we calculated the ratios as channel 1 over channel 2 in both (i.e. the dye-swapped slide was not treated as a dye swap). In this case, the expression levels should be exact opposites of each other. Table AII is the DUMB table for this comparison with channel sum 1000 and fold threshold 1.4 for both arrays. All the parameters of agreement were considerably lower than in previous comparisons. Of particular note are the NDU and both kappa statistics. This example also demonstrated that the channel sum filter and fold-change criteria can be adjusted in a manner that will increase the apparent agreement. Since the dye-swapped slide was not treated properly in this case, higher numbers in the DU and UD cells were expected. Adjusting the channel sum filter eliminated these expected differences. As the channel sum filter was lowered, the differences became more obvious. However, this is not a shortcoming of the table; instead it reinforces the need to examine the parameters of agreement rather than relying solely on the values of the cells.

Table AII
figureTabAII

Down-Up-Missing-Below (DUMB) table generated from a pathological comparison (Unamplified 4 [1000] vs Unamplified 5 reversed [1000]).a In this case, a dye-swapped pair was compared, but the dye-swapped slide was not treated as a dye swap. The channel sum was 1000 and the fold-change threshold was 1.4. The parameters of agreement were all considerably lower than in previous comparisons

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Bearden, E.D., Simpson, P.M., Peterson, C.A. et al. Assessing the Reliability of Amplified RNA Used in Microarrays. Appl-Bioinformatics 5, 67–76 (2006). https://doi.org/10.2165/00822942-200605020-00001

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Keywords

  • Venn Diagram
  • Percent Agreement
  • Label Target
  • Tabular Method
  • Fold Threshold