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

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