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BioMicroCenter:Microarrays: Difference between revisions
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==== Labeling Kits ==== | ==== Labeling Kits ==== | ||
[[Image:BMC_NugenKit.png|right]] | [[Image:BMC_NugenKit.png|right]] | ||
Affymetrix | Affymetrix retired their old 3' and Exon Array kits recently. BMC evaluated several new technologies to replace the kits. The newer kits are faster and require less starting material. Based on the type of protocol, we have chosen different kits for amplifying and labeling the RNA.Information on the evaluation can be found '''[[BioMicroCenter:3primeIVT|HERE]].'''. More information about some of our labeling protocols is below. | ||
* '''Prokaryote arrays''': For both Agilent and Affymetrix, we have had success using the [[http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=339 Ambion MessageAmp II-Bacteria kits]. These kits require 100ng of total RNA to start with. The RNA is then polyadenylated. The polyA RNA is then reverse transcribed with a T7 containing primer and 2nd strand of DNA is created. The T7 primer is then used to make antisense cRNA incorporating biotinylated or Cy3/Cy5 nucleotides which are hybridized to the arrays. We can use the old Affymetrix labeling system by request. | |||
* '''Affymetrix 3' arrays | Agilent Arrays (option1)''': For standard Affymtrix arrays and for some Agilent arrays we have been using the NuGEN Applause system. This system uses 50-100ng of total RNA as input. A unique feature of the NuGEN kits is that they use cDNA for hybridization instead of cRNA. Several studies have found increased specificity with the NuGEN system(eg. Eklund et al., Nat.Biotech. 2006). Preliminary tests were performed using samples from the White lab and analyzed in collaboration with Charlie Whittaker at the Koch Institute Bioinformatics and Computing Core Facility. The NuGEN kits showed significantly higher sensitivity then the Affymetrix kits coupled with decreased levels of background hybridization. All of the kits showed highly similar changes in expression between two samples, however the absolute level of detected transcripts were quite different. NuGEN kits can be adapted for Agilent arrays much the same way the Ambion prokaryote kits can. | |||
NuGEN | * '''Affymetrix Exon Arrays ''': For whole transcriptome analysis, we have two kits we are currently evaluating. We have the most experience with the NuGEN wt-ovation Exon kit, which works much the same way as the 3' kit does. We are also evaluating the Ambion exon kit which is recommended by Affymetrix. Standard kits for both can handle samples over 50ng. The NuGEN kit is unique in that [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-pico/ it can handle samples down to 100pg using their Pico kit]. NuGEN also sells a [[http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-one-direct/ single cell] preparation kit. Please contact [[BioMicroCenter:People|Stuart Levine]] if you are interested in trying out these technologies. | ||
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. | |||
== QUALITY CONTROL == | == QUALITY CONTROL == | ||
