Gene Expression Analysis

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.