1,372
edits
BioMicroCenter:Microarrays: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
no edit summary
No edit summary |
|||
| Line 43: | Line 43: | ||
|- | |- | ||
| CGH | | CGH | ||
| | | 3 μg (DNA) | ||
| 6 μL | | 6 μL | ||
| Agilent | | Agilent | ||
| Line 49: | Line 49: | ||
|- | |- | ||
| small RNA | | small RNA | ||
| | | 1 μg | ||
| 6 μL | | 6 μL | ||
| Agilent | | Agilent | ||
| Line 63: | Line 63: | ||
<br> | <br> | ||
=== TURN AROUND === | === TURN AROUND === | ||
The BioMicro Center will turn around your samples as quickly as possible | The BioMicro Center will turn around your samples as quickly as possible. Currently the average turn around time is 1-2 weeks. | ||
<br> | <br> | ||
== ARRAY SELECTION == | == ARRAY SELECTION == | ||
There are several considerations to take into account in selecting your microarray. We provide support for multiple array platforms from two different manufacturers (Agilent and Affymetrix). Additionally, multiple labeling methods are available for each platform. Each labeling method has different sample requirements and multiple labeling methods should NOT be combined in doing post-hybridization analysis. | There are several considerations to take into account in selecting your microarray. We provide support for multiple array platforms from two different manufacturers (Agilent and Affymetrix). Additionally, multiple labeling methods are available for each platform. Each labeling method has different sample requirements and multiple labeling methods should NOT be combined in doing post-hybridization analysis. The BioMicro Center staff is available to discuss the different options available and to assist you in experimental design. | ||
=== Agilent vs. Affymetrix === | === Agilent vs. Affymetrix === | ||
[http://www.affymetrix.com AFFYMETRIX] arrays are very high density arrays of 25mer 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.affymetrix.com AFFYMETRIX] arrays are very high density arrays of 25mer 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. | [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. Because of the lower probe density and the longer oligos used, each probe is evaluated separately. [[Image:Multiple_arrays.jpg|right]] Each glass slide can contain multiple identically-sized arrays (2, 4, or 8), reducing the cost per hybridization. Our Agilent scanner (Donated by the Bell lab) was recently upgraded (April 2011) and can handle Agilent arrays of up to 1 million probes per slide. The new scanner is capable of scanning with 2, 3, 5 or 10 micron resolution, identifying weaker signals and preventing feature saturation. Typical mammalian expression arrays are in a 4x44k or 8x60k format where each slide contains four or eight identical arrays that are separable 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. | ||
'''As of July 2011, on a per sample basis, Agilent arrays now cost considerably less then Affymetrix Genechips (under $400 per array), but must be processed as whole slides.''' | |||
Please note that the BioMicro Center no longer supports printing of microarrays in house. 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 microarray printing, 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 | |||
=== Labeling Methods === | === Labeling Methods === | ||
| Line 84: | Line 82: | ||
==== Labeling Kits ==== | ==== Labeling Kits ==== | ||
[[Image:BMC_NugenKit.png|right]] | [[Image:BMC_NugenKit.png|right]] | ||
Affymetrix retired their old 3' and Exon Array kits | Affymetrix retired their old 3' and Exon Array kits in 2009. 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. | * '''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 | * '''Agilent Arrays (option 1)''': For Agilent expression, CGH and miRNA arrays our primary labeling kits are purchased directly from Agilent. | ||
* '''Affymetrix arrays | Agilent Arrays (option 2)''': For standard Affymtrix arrays and for some Agilent arrays we have been using the NuGEN Applause and Ovation-WT systems. 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. | |||
* ''' | * '''Low Concentration Samples''': For very low concentration samples, the BioMicro Center uses the NuGEN [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-pico/ Pico kit]that can handle inputs down to 500pg (~50 cells). NuGEN also sells a [[http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-one-direct/ single cell] preparation kit. The Pico kit can be used on Agilent, Affy 3' and Affy exon microarrays. | ||
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. | 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. | ||
| Line 146: | Line 146: | ||
=== Agilent Data Analysis === | === Agilent Data Analysis === | ||
Agilent Scanner is available to members of the MIT Community for $50 per scan. We are currently running Feature extractor version | Agilent Scanner is available to members of the MIT Community for $50 per scan. We are currently running Feature extractor version 10.5. For questions about equipment, contact [[BioMicroCenter:People|Manlin Luo]] 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 == | == LINKS == | ||
