Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance

Abstract

Background: A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20-200 micro-grams total RNA or 0.5-2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material.

Results: Using analysis of variance (ANOVA) and multiple hypothesis testing, we estimated the impact of amplification on the preservation of gene expression ratios. Both methods showed that the gene expression ratios were not completely preserved between amplified and non-amplified material. We also compared the expression ratios between the two cell lines for the amplified material with expression ratios between the two cell lines for the non-amplified material for each gene. With the aid of multiple t-testing with a false discovery rate of 5%, we found that 10% of the genes investigated showed significantly different expression ratios.

Conclusion: Although the ratios were not fully preserved, amplification may prove to be extremely useful with respect to characterizing low expressing genes.

Figure 1

Experimental design. For a particular cell line, the material was divided into six batches; two batches of non-amplified RNA were directly labeled (white shapes) and four batches of diluted total RNA were amplified before labeling (grey shapes). These batches were split in two and labeled Cy5 (red) or Cy3 (green). In total, four array experiments were based on non-amplified material, and eight experiments were based on amplified material.

Figure 2

(A) Plot of the difference in log₂-ratio between the amplified and the non-amplified material for the log₂-ratio of the two cell-lines (MT-1, U2OS) vs. the p-value for each of the 4331 genes, calculated for hypothesis testing. (B) Plot of the average log₂-ratio of the two cell-lines (MT-1 and U2OS), for the amplified and the non-amplified material for each of the 4331 genes investigated during hypothesis testing. The genes in the "conservative set" are in green, the genes in the "rejected set" are in red and the genes in the "undetermined set" are in black.