No, there is no set maximum distance. However, strong preference is given to probes closer to the 3′ end.
Illumina recommends quantitation of amplified RNA by fluorometry using RiboGreen reagent and additional qualitative analysis with the Agilent Bioanalyzer or by electrophoresis through agarose gel. Measuring A260 absorbance with a spectrophotometer offers a less precise RNA quantitation method.
Use at least 50 ng total RNA. This input generally produces 5–10 ug of amplified RNA, which is sufficient for 10–20 arrays.
No. The low variability and high inter- and intra-slide reproducibility of Illumina arrays make it unnecessary to perform dual-color experiments.
Two kits have been tested for use with Illumina gene expression products:
The Ovation Amplification Kits from NuGen have also demonstrated acceptable results with the use of a modified protocol. (Illumina does not provide technical support for these kits.)
p = 10'(DiffScore*sgn(µcond-µref)/10)
Illumina has found quantification by RiboGreen to be more accurate than spectrophotometer readings. Contaminants from purification columns can give artificially high readings. If you must use A260, sample measurements should be > 0.5 to minimize the impact of contaminants on amplification.
Illumina has had poor results when using fragmented sample on our arrays. The Affymetrix protocol requires fragmentation during sample preparation due to the short probes used on their arrays as it reduces the secondary structure of RNA. Illumina's longer probes allow for more stringent hybridization conditions which preclude the need for RNA fragmentation. If you have labeled sample which has not been fragmented, you might obtain satisfactory results, depending on the age and quality of the sample.
Descriptions for all of the column headers can be found in the document Bead Manifest Field Descriptors located on the documentation CD included in the startup kit.
Illumina has not tried any two-round amplification kits. However, some customers have reported successful use of two-round IVT kits with Illumina gene expression arrays.
Illumina has tested only RNA isolated using RNEasy from Qiagen, one of the most frequently used methods. We do not anticipate a major impact on performance with other appropriate, well-established isolation techniques.
Sensitivity is 1 in 250,000.
Expect 1.35 fold.
Yes. Our probes (human and mouse) have been mapped to Ensembl. The Illumina probe track can be turned on by checking the box next to Illumina Probes on the DAS Sources dropdown menu in Contigview. When the page refreshes, blue features indicate the location of probes.
The GFF text files giving the mapping information for probes to mouse and human can be downloaded from the Sanger Institute website.