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BioMicroCenter:Microarrays: Difference between revisions
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=== Agilent vs. Affymetrix === | === Agilent vs. Affymetrix === | ||
[http://www.affymetrix.com AFFYMETRIX] arrays are very high density arrays of 30mer oligonucleotides built by lithography. An Affymetrix probeset is composed of numerous neighboring perfect match and mismatch probes that are combined and used in the calculation of intensity and relative expression. Affymetrix arrays are the most common arrays used in genomics right now and have a rich bioinformatic architecture that allows for a large number of analyses. While expression arrays are the most common, Affymetrix also creates arrays for SNP calling and ChIP-chip. Affymetrix expression arrays come in two flavors: 3' and exon. 3' arrays use oligo dT priming while exon arrays amplify from the whole RNA using random primers. Affymetrix arrays are one-color arrays and use biotinylated nucleotides in the cRNA/cDNA to recruit HRP-peroxidase to the probes. Affymetrix arrays come as single arrays from a broad list of species. The BioMicro Center has a hybridization oven, two 450 fluidics devices and a 7G scanner for handling Affymetrix arrays. | |||
[http://www.agilent.com AGILENT] arrays are lower density arrays then Affymetrix, but have much longer oligos (60mers) and are printed on glass slides using dot-matrix printing technologies. Because this method does not require the creation of metal plates, novel microarray designs cost no more then designs created by Agilent. Agilent arrays can be used for a variety of assays including gene expression, ChIP-chip, miRNA hybridization, and CGH. Our Agilent scanner can handle Agilent arrays of up to 250k probes per slide (Donated by the Bell lab). Because of the lower probe density and the longer oligos used, each probe is evaluated separately. Each glass slide can contain multiple identical arrays (2, 4, or 8), reducing the cost per hybridization, often below the level of Affymetrix arrays. Typical mammalian expression arrays are in a 4x44k format where each slide contains four identical arrays that are seperable by a gasket slide. Agilent arrays can be hybridized either as one-color or two-color using Cy5 and Cy3 labeled cRNA/cDNA. We do not currently maintain a stock of Agilent arrays but do have a significant discount on the purchase of arrays. | |||
Please note that the BioMicro Center no longer supports self printed arrays. Custom oligonuclotide arrays are available from Agilent at the same cost as their commercial arrays using their [http://www.chem.agilent.com/en-US/products/instruments/dnamicroarrays/pages/gp50660.aspx eArray system] (we can [[BioMicroCenter:Computing#BioInformatics_Services|help you]] through the process of designing arrays). For anyone requiring self printed arrays, we encourage you to contact [http://jura.wi.mit.edu/genomecorewiki/index.php/Tom_Volkert Tom Volkert at the Whitehead Institute CGT]. | Please note that the BioMicro Center no longer supports self printed arrays. Custom oligonuclotide arrays are available from Agilent at the same cost as their commercial arrays using their [http://www.chem.agilent.com/en-US/products/instruments/dnamicroarrays/pages/gp50660.aspx eArray system] (we can [[BioMicroCenter:Computing#BioInformatics_Services|help you]] through the process of designing arrays). For anyone requiring self printed arrays, we encourage you to contact [http://jura.wi.mit.edu/genomecorewiki/index.php/Tom_Volkert Tom Volkert at the Whitehead Institute CGT]. | ||
== | === Labeling Methods === | ||
=== Labeling Kits === | ==== Labeling Kits ==== | ||
[[Image:BMC_NugenKit.png|right]] | [[Image:BMC_NugenKit.png|right]] | ||
Affymetrix is retiring their old 3' Array kits in September 2009. In response, BMC has been evaluating new technologies. The newer kits are faster and require less starting material. We have selected the NuGEN technology as the primary labeling method we will be using going forward for eukaryotic arrays because of their ease and the modularity of their system that allows us to combine the processing of Affymetrix 3', Affymetrix Exon, and Agilent arrays. Information on the evaluation can be found '''[[BioMicroCenter:3primeIVT|HERE]].'''. We DO NOT RECOMMEND mixing labeling types within an experiment as we have observed large differences between the absolute intensities of signals between labeling methods. Arrays can still be hybridized using the Affymetrix 3'IVT, Affymetrix Exon and old Affymetrix 3' array by request (please ask for pricing). | Affymetrix is retiring their old 3' Array kits in September 2009. In response, BMC has been evaluating new technologies. The newer kits are faster and require less starting material. We have selected the NuGEN technology as the primary labeling method we will be using going forward for eukaryotic arrays because of their ease and the modularity of their system that allows us to combine the processing of Affymetrix 3', Affymetrix Exon, and Agilent arrays. Information on the evaluation can be found '''[[BioMicroCenter:3primeIVT|HERE]].'''. We DO NOT RECOMMEND mixing labeling types within an experiment as we have observed large differences between the absolute intensities of signals between labeling methods. Arrays can still be hybridized using the Affymetrix 3'IVT, Affymetrix Exon and old Affymetrix 3' array by request (please ask for pricing). | ||
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We have also switched our prokaryotic labeling system from the Affymetrix system to one developed by [http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=339 Ambion]. The Ambion system requires significantly less input RNA and has a more streamlined protocol. The Affymetrix labeling system is still available by request. | We have also switched our prokaryotic labeling system from the Affymetrix system to one developed by [http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=339 Ambion]. The Ambion system requires significantly less input RNA and has a more streamlined protocol. The Affymetrix labeling system is still available by request. | ||
=== Small Cell Numbers === | ==== Small Cell Numbers ==== | ||
NuGEN has recently developed kits that allow the analysis of very [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-pico/ small amounts of purified RNA (100pg)] or even [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-one-direct/ single cells]. Please contact [[BioMicroCenter:People|Stuart Levine]] if you are interested in trying out these technologies. | NuGEN has recently developed kits that allow the analysis of very [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-pico/ small amounts of purified RNA (100pg)] or even [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-one-direct/ single cells]. Please contact [[BioMicroCenter:People|Stuart Levine]] if you are interested in trying out these technologies. | ||
== | |||
== QUALITY CONTROL == | |||
=== RNA === | |||
=== Affymetrix Arrays === | |||
=== Agilent Arrays === | |||
== DATA ANALYSIS == | |||
=== Affymetrix Data Analysis === | === Affymetrix Data Analysis === | ||
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After you have completed your scan, please make sure to sign the scanner record form, which is near the equipment and to remove your files from the computer. CD-Rs are available if needed. | After you have completed your scan, please make sure to sign the scanner record form, which is near the equipment and to remove your files from the computer. CD-Rs are available if needed. | ||
== LINKS == | |||
=== [[BioMicroCenter:Pricing|PRICES]] === | |||
=== [[BioMicroCenter:FAQ#NON_MIT_USERS|Information for non-MIT labs]] === | |||
=== [[BioMicroCenter:FAQ#HOW_DO_I_GET_MY_DATA|Obtaining your results]] === | |||
You will be notified by email that your data is ready. At that time please pick up any leftover samples and arrays belonging to you. Samples will be held at the facility at -80C for 60 days after your data is generated. Information on downloading your data is in the link above. | |||
