Statistical evaluation of transcriptomic data generated using the Affymetrix one-cycle, two-cycle and IVT-Express RNA labelling protocols with the Arabidopsis ATH1 microarray.
BACKGROUND
Microarrays are a powerful tool used for the determination of global RNA expression. There is an increasing requirement to focus on profiling gene expression in tissues where it is difficult to obtain large quantities of material, for example individual tissues within organs such as the root, or individual isolated cells. From such samples, it is difficult to produce the amount of RNA required for labelling and hybridisation in microarray experiments, thus a process of amplification is usually adopted. Despite the increasing use of two-cycle amplification for transcriptomic analyses on the Affymetrix ATH1 array, there has been no report investigating any potential bias in gene representation that may occur as a result.


RESULTS
Here we compare transcriptomic data generated using Affymetrix one-cycle (standard labelling protocol), two-cycle (small-sample protocol) and IVT-Express protocols with the Affymetrix ATH1 array using Arabidopsis root samples. Results obtained with each protocol are broadly similar. However, we show that there are 35 probe sets (of a total of 22810) that are misrepresented in the two-cycle data sets. Of these, 33 probe sets were classed as mis-amplified when comparisons of two independent publicly available data sets were undertaken.


CONCLUSIONS
Given the unreliable nature of the highlighted probes, we caution against using data associated with the corresponding genes in analyses involving transcriptomic data generated with two-cycle amplification protocols. We have shown that the Affymetrix IVT-E labelling protocol produces data with less associated bias than the two-cycle protocol, and as such, would recommend this kit for new experiments that involve small samples.
Centre for Plant Integrative Biology, University of Nottingham, Nottingham, LE12 5RD, UK.
Tara J
TJ
Holman
Centre for Plant Integrative Biology, University of Nottingham, Nottingham, LE12 5RD, UK.
Michael H
MH
Wilson
Centre for Plant Integrative Biology, University of Nottingham, Nottingham, LE12 5RD, UK.
Kim
K
Kenobi
Centre for Plant Integrative Biology, University of Nottingham, Nottingham, LE12 5RD, UK.|School of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.
Ian L
IL
Dryden
Centre for Plant Integrative Biology, University of Nottingham, Nottingham, LE12 5RD, UK.
T Charlie
TC
Hodgman
Centre for Plant Integrative Biology, University of Nottingham, Nottingham, LE12 5RD, UK.|School of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.
Andrew Ta
AT
Wood
Centre for Plant Integrative Biology, University of Nottingham, Nottingham, LE12 5RD, UK.|Department of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Nottingham, LE12 5RD, UK.
Michael J
MJ
Holdsworth