The Illumina BeadChips described below are available for service at GPCF - Information as provided by Illumina Inc.
The Sentrix BeadChip technology is set up to perform multiple hybridizations in parallel. The Sentrix-12 chip type is most economic with 12 samples (or multiple thereof).
HumanHT-12 v4 Expression BeadChip Kits
The BeadChip HumanHT-12 v4 offers comprehensive analysis of genome-wide expression on a single array.
- probes are defined gene-specific 50mer oligonucleotides
- ca. 300,000 accessible oligonucleotides per bead/spot
- on average 15x redundancy for each transcript
- Provides comprehensive coverage of the transcribed human genome on a single array
- Analyzes the expression level of 31,000 annotated genes with more than 47000 probes derived from the NCBI RefSeq Release 38 (November 7, 2009) and other sources.
- comprised of more than 7,000,000 beads per array, meaning 12x7,000,000 beads per chip
- Up-to-date gene list and annotation of HumanHT-12 v4
For more information please download the HumanHT-12 v4 Whole Genome BeadChip datasheet from Illumina. One physical array is made up of 12 identical, but independent chips.
Information about the technology employed by Illumina is primarily taken from publications of the company.
Figure 1: Schematic view of a bead coupled with an oligonucleotide, consisting of the address code and a 50 base gene-specific sequence
In detail, Illuminaâs BeadArrayâ¢ technology uses silica microspheres (beads) as the array elements. Each microsphere of a diameter of 3 Âµm, is derivatized with a particular oligonucleotide that acts as a probe for the complementary sequence in an assay solution (Steinberg et al., 2004). The oligonucleotides synthesized are >72 bases, consisting of an address code and a 50 base gene-specific sequence. Each oligonucleotide is tightly quality controlled and coupled to a batch of beads (see figure 1).
Each bead carries >1x105 identical >72mer oligonucleotides. For a 47k gene expression array, 47,000 bead types are prepared, and equal aliquots of each type are pooled. The pool is spread across prefabricated microarrays with defined microwells that fit to the bead size (see figure 2). The beads are immobilized within the cavities and the 25 base addresses are decoded, to allocate each bead to the respective gene sequence (Gunderson et al., 2004). The error rate in decoding is considered low (<1x10-4).
Each bead type has an average 30x representations on the chip â a strategy that provides the statistical accuracy of multiple measurements.
- The total mature RNA is isolated from the cell/tissue being studied. This RNA has already been âprocessedâ (removal of the noncoding introns and splicing together of the coding exon) as well as the addition of a poly-A tail
- The RNA is turned into a double stranded DNA copy known as a 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 uracil bases tagged with the Biotin.
- The Biotin-labeled cRNA is then added to the array
- Anywhere on the array where a RNA fragment and an oligonucleotide on a bead are complimentary, the RNA sticks to the probe on the bead
- 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
- 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.
Seven control categories are built into the Illumina BeadChip system, six of which are being used by GPCF. These cover every aspect of an array experiment from the biological specimen, to sample labeling, to hybridization and signal generation. We automatically track performance of these controls and generate a report for each array.
Controls for the Biological Specimen (Housekeeping Controls): The intactness of the biological specimen can be monitored by housekeeping gene controls. These controls consist of probes to housekeeping genes, two probes per gene that should be expressed in any cellular sample.
Controls for Sample Labeling (RNA spike): These controls are NOT used at GPCF. These controls consist of four probes corresponding to artificial polyadenylated spike RNAs (Bacillus subtilis). The RNAs must be spiked into each sample immediately preceding the reverse transcription step. These spike RNAs are amplified and labeled in the same reaction as the sample, and thus act as tracers for reaction success.
|Cy3-Labeled Hyb Controls: These controls consist of six probes with corresponding Cy3-labeled oligonucleotides present in the hybridization buffer. Following successful hybridization, they produce a signal independent of both the cellular RNA quality and success of the sample prep reactions. Target oligonucleotides for the Cy3 Hyb controls are present at three concentrations (low, medium, and high), yielding gradient hybridization response.|
|Low Stringency Hyb Control: This control category contains four probes, corresponding to the medium- and high-concentration Cy3 hyb control targets. In this case, each probe has two mismatch bases distributed in its sequence. If stringency is adequate, these controls yield very low signal. If stringency is too low, they yield signal approaching that of their perfect match counterparts in the Cy3 hyb control category.|
|High Stringency Hyb Control: This control consists of one probe corresponding to a Cy3-labeled oligonucleotide target. The probe/target sequences have a very high G+C content, and should thus hybridize even if hybridization stringency is too high. Hybridizations with too-high stringency are detected on the basis of a signal present from this control in the absence of signal from the other hybridization control probes.|
|Signal Generation Controls (Biotin Control): This category consists of two probes with complementary biotin-tagged oligonucleotides present in the Hyb E1 buffer. Successful secondary staining is indicated by a positive hybridization signal from these probes.|
|Negative Controls: This category consists of 800-1600 probes of random sequence selected to have no corresponding targets in the genomes. The mean signal of these probes defines the system background. This is a comprehensive measurement of background, representing the imaging system background as well as any signal resulting from non-specific binding of dye or cross-hybridization. We use the signals and signal standard deviation of these probes to establish gene expression detection limits.|