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.
Direct Hybridization (Direct Hyb) refers to the direct hybridization of probes on the array to the complementary cRNA molecule in your sample. This is in contrast to Illumina's flagship GoldenGate® Genotyping Assay, which incorporates a unique oligonucleotide onto the sample sequence. This unique oligonucleotide then hybridizes to the complementary probe on the array.
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.
Automation of the DASL® Assay is available. However, Illumina currently does not offer automation for the Direct Hyb protocol. Contact Ambion for information about automation of the TotalPrep protocol.
The intensity range over which a fold change of ~1.35 is significantly distinguishable is greater than 3 logs.
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.
A = All isoforms. The probe is designed to hit all splice isoforms of a gene.
I = Isoform specific. The probe is designed to hit a specific splice isoform of a gene, for which multiple isoforms are known to exist.
S = Single isoform. The gene has only one known splice isoform and our probe hits it.
M = Multiple isoforms. This gene has multiple isoforms. The probe targets more than one and fewer than all of them.
The probe design and hybridization conditions were optimized for use with amplified cRNA. Varying the sample to cDNA would require re-optimization of every step of the protocol’s hybridization and wash conditions. Our protocol does not include a pre-block step of the microarray before adding sample. Adding Cy3-labeled sample would produce high background and poor results.
Yes, this is known as bead-level data. Contact your Field Application Scientist (FAS) or Technical Support Representative for assistance.
Sensitivity is 1 in 250,000.
Expect 1.35 fold.
It should not be necessary to remove rRNA before labeling. The ribosomal RNA does not get amplified in the protocol and should represent a very small percentage of the final product.
Illumina has not tested kits designed to label microbial RNA. Reagent vendors such as Ambion® do sell kits for this application. These kits may work, but there is no particular kit that we recommend. It is important that a single source of biotin-16-UTP (i.e., same vendor) is used for all labeling reactions (e.g., Ambion #8452 or #8453).
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. Clicking on a probe opens up a floating menu with probe information and a link to a page giving more information about the probe. The GFF text files giving the mapping information for probes to mouse and human can be downloaded from the Sanger Institute website ( http://www.sanger.ac.uk/Software/ formats/GFF/): - Human GFF file (right-click and select Save As) - Mouse GFF file (right-click and select Save As) More information about the GFF data format with an explanation of the fields can be found at: http://www.sanger.ac.uk/Software/formats/GFF/
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.