Affymetrix GeneChip® expression analysis
arrays enable whole-genome expression profiling in several model organisms.
For each gene represented on an array, multiple probe pairs (e.g. 11 pairs),
chosen from the 3´end of the respective mRNA sequence, are synthesized
on the array. Each probe pair consists of a Perfect Match probe (PM - perfectly
complementary to a target sequence) and the Mismatch probe (MM – identical
to PM except for a single base mismatch in its center) allowing the quantitation
and subtraction of signals caused by non-specific cross-hybridization (illustration
of principle).
The sample or target to be analyzed on a GeneChip®array
is prepared from total RNA isolated from the tissues or cells under study
(simplified
graphical overview). In a first step, double-stranded cDNA is generated
that carries a T7 promoter at its 5´end. This promoter is then used
for in vitro transcription, in which biotinylated nucleotides (biotin-ddUTP
and biotin-ddCTP) are incorporated into the resulting cRNA. This second step
leads to an approximate 100-fold linear amplification of the starting material,
thus allowing the starting material needed to be as little as five micrograms
of total RNA for the standard protocol. A new small sample protocol does
not incorporate biotinylated nucleotides into the cRNA in this step, but
instead uses the non-labeled cRNA for a second round of cDNA production and
in vitro transcription with biotinylated nucleotides. In this protocol
the amount of starting material can be reduced to as low as 50 ng of total
RNA, facilitating studies in which the starting material is the limiting
factor, such as samples from laser capture microdissections, small biopsies,
and flow-sorted cells. In both protocols, the biotin-labeled cRNA is then
fragmented into 35-200 bases fragments by metal-induced hydrolysis and hybridized
to GeneChip® arrays for 16 hours. Unlike spotted cDNA arrays
the two samples to be compared are not hybridized to the same array, but
each sample is separately hybridized to a single GeneChip®
array. The bound biotinylated cRNA fragments are labeled with a fluorescent
streptavidin conjugate and fluorescent intensities for each probe cell are
acquired on a GeneChip® scanner.
As a first step of the analysis, arrays are normalized to reduce variation of non-biological origin between them. Then, using standard statistical techniques and the fluorescent intensities of all probe pairs representing a gene on the array, a signal value is calculated, which assigns a relative measure of abundance to the transcript. In addition, a detection call is calculated that indicates whether a transcript is reliably detected (Present) or not detected (Absent). In the subsequent comparison analysis the expression values of each gene in the two samples are compared with each other to identify genes up- or down-regulated and to quantify these changes.
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