The following Affymetrix GeneChipsÂ® are used at GPCF (Information as provided by Affymetrix).
|Array type||3' IVT expression cartridge||Gene array|
|Assay||3' end of transcript||Whole-transcript sense strand|
The novel Clariom S assays focus on well-annotated genes (>20,000), providing researchers with the ability to perform cost-effective gene-level expression profiling studies and to quickly assess changes in key genes and pathways. For more details click here.
- Provides comprehensive coverage of the transcribed human genome on a single array
- Analyzes the expression level of over 47,000 transcripts and variants, including 38,500 well-characterized human genes
- Comprised of more than 54,000 probe sets and 1,300,000 distinct oligonucleotide features
- Up-to-date gene list and annotation of U133Plus 2.0 (>20MB!)
For more detail download the Human U133Plus 2.0 data sheet.
- Provides coverage of the transcribed mouse genome on a single array
- 45,000 probe sets analyze the expression level of over 39,000 transcripts and variants from over 34,000 well characterized mouse genes
- Up-to-date gene list and annotation of Mouse 430 2.0 (>15MB!)
For more detail download the Mouse 430 2.0 data sheet.
- Provide comprehensive coverage of the transcribed rat genome
- 31,000 probe sets analyze the expression level of over 30,000 transcripts and variants from over 28,000 well-substantiated rat genes
- Up-to-date gene list and annotation of Rat 230 2.0 (>10MB!)
For more detail download the Rat 230 2.0 data sheet.
Information about the technology employed by Affymetrix is primarily taken from publications of the company.
The Affymetrix technology uses photolithographic synthesis of oligonucleotide on microarrays. Standard GeneChips are ~1.6 cm2 in size. These chips can hold up to 1.6 million features (spots). One feature is composed of a large number of identical oligonucleotides of 25 bases that are synthesized onto the chip as described in figure 1. For more detailed information, please download a respective presentation from Affymetrix.
The synthesis of these oligonucleotides on GeneChip microarrays are based on the concept of photolithography
- Light is shined through a mask onto a chip that has initial starting strands where the DNA will be built from
- The mask has specific tiny openings that allow the light to come in contact with the wafer at specific sections (in this diagram there are 5 probes only and each could represent a different feature)
- Any place where light hits, removes a âprotectiveâ group from the strands
- Free nucleotides (the red T) are washed over the chip and the nucleotides will combine with any strand that had lost itsâ protective group in the previous step
- This is then repeated (shine light through a mask, deprotect the strands, add free nucleotides) numerous times until each strand built is 25 base pairs long
The presence of messenger RNA (mRNA) is detected by a series of probe pairs that differ in only one nucleotide. Hybridization of fluorescent mRNA to these probes pairs on the chip is detected by laser scanning of the chip surface.
Affymetrix GeneChips are packed in cartridges (figure 2), which protect the chip itself. In addition, the cartridge serves as reaction chamber for the hybridization.
The middle part of figure 2 is image of an actual expression array after scanning. It describes the full array, with every feature represented. The right part of the image on the right represents a magnified view of a section of the array, showing only 324 features. Each feature is scanned for its' intensity and it is evident that there is a wide range of intensities in this area. The black features represent no intensity (no RNA hybridized to the respective probe in the feature). The intensity level from lowest to highest by color is: Dark blue -> Blue -> Light Blue -> Green -> Yellow -> Orange -> Red - > White.
More intensity means more RNA bound to a specific feature, which basically means the gene was expressed at a higher level.
Figure 3 below describes the workflow of Affymetrix experiment.
Labeling procedure for 3Â´-IVT Arrays
- The total mature RNA (cytoplasmic RNA) is isolated from the tissue/cell being studied.
- The RNA is turned into a double stranded cDNA. This is done through reverse transcription. This is done because RNA itself is not a very stable molecule and the cDNA is a way to store the RNA for a much longer period of time
- When it comes time to run the array, the cDNA is allowed to go through in vitro transcription back to RNA (now known as cRNA), but this RNA is labeled with Biotin. This is done by having all the uracil bases tagged with the Biotin.
- This labeled cRNA is then randomly fragmented in to pieces anywhere from 30 to 400 base pairs in length
- The fragmented, Biotin-labeled cRNA is then added to the array
- Anywhere on the array where a RNA fragment and a probe are complimentary, the RNA hybridizes to the probes in the feature.
- The array is then washed to remove any RNA that is not stuck to an array (i.e., no match was made) and then stained with the fluorescent molecule that sticks to Biotin (Cy5 conjugated to streptavidin)
- Lastly, the entire array is scanned with a laser and the information is kept in a computer for quantitative analysis of what genes were expressed and at what approximate level