The most commonly used 3�� In Vitro Transcription (3��IVT) Affymetrix microarrays consist of probesets usually incorporating 11 Perfect-Match (PM) 25 nucleotide (nt)-long oligonucleotide probes specific to a ~600 nt region of the transcript’s 3��-UTR and an additional set of corresponding mismatch (MM) probes where the central 13th nucleotide is replaced with its complementary equivalent used for the accession of non-specific binding strength. The next generation of Whole Transcript (WT) expression analysis arrays (like the HuGene-1_0-ST) utilize a set of background intensity probes that have no homology to the transcripts of the organism analyzed which are used to estimate the non-specific binding based on the varying GC content of the probes (the number of G and C nucleotides in the sequence).

Additionally, the probes are selected based on various gene exons and not only on the 3��-UTRs as in older designs, which allows more precise separation of the intensities for various splice variants. Exon-specific probesets usually comprise four probes, although transcript- or gene-specific sets include on the average over 25 probes (HuGene-1_0-ST), significantly exceeding the probe numbers in older designs. Due to significant differences between both platforms, various approaches to the data analysis are required. Additionally, the platforms vary in the sample preparation procedures knowledge of which is required for the appropriate understanding of the data analyzed.

The basic steps of a microarray experiment include RNA isolation, cDNA synthesis, amplification and labeling, cRNA fragmentation, hybridization, washing and staining and finally a complete surface scan of the microarray. The main differences Anacetrapib between WT and 3�� IVT microarrays concern the cDNA synthesis step and result from the need to either achieve a high quality whole transcript amplification or amplification of the 3��-UTR region. 3��IVT microarrays utilize the oligo(dT) primer which, by binding to the 3��UTR region, initiates the cDNA synthesis in the 3��->5�� direction. This approach allows to achieve a very high yield of amplification in the close vicinity of the 3�� region although it is very susceptible to RNA degradation [2]. In contrast, WT microarrays are based on random primers which can attach to various regions of the transcript thereby promoting the cDNA synthesis reaction independently from the 3�� region. Both primers include a T7 polymerase promoter which in the amplification process leads to a significant increase in the amount of target material due to the in vitro transcription process. This step produces cRNAs whose sequence is complementary to the isolated RNA molecules.