Microarray analysis after RNA amplification can detect pronounced differences in gene expression using limma

TLDR

RNA amplification is required to profile gene expression from small tissue samples, but T7‑based techniques, while reproducible, can distort true target abundance. The study aimed to determine how such distortions affect the detection of biological variation in gene expression and to delineate the usability limits of amplification methods. The distortion occurs when a shift in one sample cannot be reflected in the other because the resulting intensity lies outside the scanner’s dynamic range. Amplification with the Affymetrix small‑sample protocol v2 reduces expression ratios and statistical significance, yet limma preserves a higher overlap of significant genes—up to 87 % of the most significant genes—making amplified microarray analysis most reliable for large expression differences.

Abstract

RNA amplification is necessary for profiling gene expression from small tissue samples. Previous studies have shown that the T7 based amplification techniques are reproducible but may distort the true abundance of targets. However, the consequences of such distortions on the ability to detect biological variation in expression have not been explored sufficiently to define the true extent of usability and limitations of such amplification techniques. We show that expression ratios are occasionally distorted by amplification using the Affymetrix small sample protocol version 2 due to a disproportional shift in intensity across biological samples. This occurs when a shift in one sample cannot be reflected in the other sample because the intensity would lie outside the dynamic range of the scanner. Interestingly, such distortions most commonly result in smaller ratios with the consequence of reducing the statistical significance of the ratios. This becomes more critical for less pronounced ratios where the evidence for differential expression is not strong. Indeed, statistical analysis by limma suggests that up to 87% of the genes with the largest and therefore most significant ratios (p < 10e-20) in the unamplified group have a p-value below 10e-20 in the amplified group. On the other hand, only 69% of the more moderate ratios (10e-20 < p < 10e-10) in the unamplified group have a p-value below 10e-10 in the amplified group. Our analysis also suggests that, overall, limma shows better overlap of genes found to be significant in the amplified and unamplified groups than the Z-scores statistics. We conclude that microarray analysis of amplified samples performs best at detecting differences in gene expression, when these are large and when limma statistics are used.