An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

Abstract

Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling.

Figure 1

Representative images of a typical TSA hybridization on the mouse 9k microarray (A) and the 95 gene, 190 element human microarray (B). In (B), duplicate spots are adjacent to each other and the consistency in hybridizations between duplicates can be observed. The image in (A) is a non-homotypic hybridization, whereas (B) is homotypic hybridization of RNA extracted from human frontal cortex.

Figure 2

Scatter plots of experiments with total RNA isolated from mouse neural stem cell cultures (neurospheres, NS). The same RNA sample was used for cDNA synthesis and labeled with either fluorescein-12-dCTP or biotin-11-dCTP. Both cDNAs were co-hybridized on the mouse 9k microarray (homotypic hybridization) and developed according to the TSA protocol provided by the manufacturer (MICROMAX; NEN). (A) The plot (on a log₁₀ scale) of Cy5 signal versus background from a representative homotypic hybridization of NS RNA on the mouse 9k array. The plots for Cy3 were similar. The points above the solid line have a signal higher than 2-fold background. The letter E indicates the signal produced by negative control spots. Notable is the clear separation between signal and background apparent in the two clusters of spots that are observed when the data are plotted in this manner. (B) A representative plot of the log₁₀ ratio (Cy3/Cy5) versus the average log₁₀ signal from both Cy3 and Cy5 channels for each spot in homotypic hybridization. It can be seen that the likelihood of false positive ratios decreases with signal strength. (C) Scatter plots of the signal generated from NS versus NS hybridizations from two experiments where the dyes were reversed. In this case, rather than labeling the same RNA, each NS culture, NS7 and NS8, was grown in separate flasks and processed separately. The clear non-linearity (curvature) produced by differences in dye incorporation and signal at different cDNA abundances can be observed.

Figure 3

Hybridization consistency on the 9K microarray using TSA amplification. In each graph, Cy5 signals are shown on the ordinate and Cy3 signals on the abscissa on a log₁₀ scale after non-linear normalization. Each dot represents the hybridization intensity of each gene. While there was more variability in hybridizations obtained from the same sample hybridized to different arrays (B) relative to the same array (A), both showed high reproducibility.

Figure 4

Comparison of RNA from frozen frontal cortex versus fixed frontal cortex. Fixed and frozen sample pairs are from an adjacent brain section from the same subject hybridized on the human array. Each dot represents the hybridization intensity of each gene. The signals for RNA from frozen tissue are shown on the ordinate in each graph on a log₁₀ scale. (A) Total RNA from frozen frontal cortex versus total RNA from formalin-fixed frontal cortex. (B) Another independent sample hybridization showing the signal obtained using total RNA from frozen frontal cortex versus total RNA from formalin-fixed frontal cortex. (C) Total RNA from frozen versus ethanol fixed frontal cortex.

Figure 5

Scatter plots of homotypic hybridization experiments using RNA isolated from the same tissue with different fixation methods. (A) Total RNA versus total RNA from frozen frontal cortex of a single subject. (B) Poly(A)⁺ RNA versus poly(A)⁺ RNA from frozen frontal cortex of a single subject. (C) Total RNA versus total RNA isolated from ethanol-fixed frontal cortex of the same subject shown in (A). (D) Total RNA versus total RNA from formalin-fixed frontal cortex of a different subject.

Figure 6

Variability across arrays and across genes. (A) Box plot of the average signal per spot across five arrays hybridized with RNA from frozen human tissue on the human array. (B) Box plot of the average log ratios across arrays. (C) Quantile–quantile plots of log ratios for two representative arrays. Although this depicts only two arrays, comparisons of the others showed a virtually identical pattern (not shown).

Figure 7

The distribution of the log ratios by gene. Ratios were generated from homotypic hybridizations of frozen tissue on five arrays. Eighty-eight genes gave signals 2-fold above background and are represented on this plot. The line depicts the average, the box encompasses the 95% confidence interval for each gene and circles lie outside the 95% confidence interval.