Global Gene Expression Assays: Quantitative Noise Analysis - Systems Biology: Networks, Models, and Applications - page 103

that takes into consideration the quantitative characterization of the noise,
and thus provides a tool for evaluating the statistical significance of gene
expression changes from different microarray experiments, is proposed.
The measurement noise is studied through replicate experiments in
which gene expression levels of a cell line are measured multiple times.
Two sources of experimental noise can be identified, starting from the
extracted mRNA, to the final readout of the gene expression levels: the
prehybridization target sample preparation steps on the one hand, and
the hybridization and subsequent readout processes (including staining
and scanning) on the other. For simplicity, these two sources of noise
will be referred to as sample preparation noise and hybridization noise.
In order to separate the noise sources due to these two factors, an
analysis will be presented of previously reported [12] multiple repli-
cate experiments where, at different stages of the experiment, the
sample is divided equally into multiple aliquots, and the subsequent
steps of the experiment are carried out independently. In this analysis
of Affymetrix GeneChips, mRNA from cells of a human Burkitt's lym-
phoma cell line (Ramos) is used for the replicate experiments. Total
RNA is extracted from the Ramos cells. The purified RNA sample is
subsequently separated equally into several subgroups. Each subgroup
independently goes through the target preparation steps, composed of
the reverse transcription (RT) step and the in vitro transcription (IVT)
step. At the end of the target sample preparation, each of the subgroups
is again split into several samples, each of which is independently
hybridized to different Affymetrix U95A GeneChip arrays. The experi-
mental design is shown schematically in figure 4.1. To have sound
Figure 4.1 Illustration of the DNA microarray replicate experiments setup.
Two different mRNA samples are used, each being probed multiple times
(replicates) with varying degrees of differences in measurement steps in order
to separate the preparation error that occurred during the reverse transcription
(RT) and in vitro transcription (IVT) processes and the final hybridization error.