BioMicroCenter:Microarrays: Difference between revisions

Revision as of 22:46, 4 June 2016

The MIT BioMicro Center has extensive experience with several different types of microarray samples and platforms. Microarray labeling and hybridization are typically performed by BioMicro Center technicians, however our scanners, hybridization ovens and wash stations are available by request. NOTE: Assisted Agilent Microarray serves are no longer available. The Agilent oven and microarray scanner is available to users who prepare their own arrays.

BioMicro Center Services

We are currently set up to primarily handle expression analysis. Our service includes:

Evaluation of sample quality using the Fragment Analyzer
Sample labeling and clean up
Array Hybridization
Data extraction and normalization

We also have discounts with different vendors and can help you purchase your arrays.

SAMPLE TYPES

The BioMicro Center has extensive experience handling samples for expression analysis.

Eukaryote	Min.RNA	Min.Vol.	Platforms	Notes
Prokaryote	1 μg	6 μL	Affymetrix	labeling using Ambion for Affymetrix arrays
3' Array (std)	50ng / 100pg	6 μL	Affymetrix	labeling using Affymetrix reagents
Exon Array	50ng / 100pg	6 μL	Affymetrix	labeling using Affymetrix reagents

SAMPLE SUBMISSION

We encourage researchers to contact us several days in advance of sample submission to allow us lead time to purchase any specialized arrays. All arrays are ordered on an as needed basis. A list of some of the available arrays and their prices can be found in this file. Information about Affymetrix arrays is available at http://www.affymetrix.com/products_services/index.affx

All submissions must be accompanied by a Affymetrix Submission Form.

TURN AROUND

The BioMicro Center will turn around your samples as quickly as possible. Currently the average turn around time is 1-2 weeks.

ARRAY SELECTION

Biomicro Center staff are available to assist you in selection of the proper microarray design. Newer Affymetrix microarrays take in to account ENCODE and RNAseq data to produce more efficient designs while older arrays have many probesets that are less useful. BioMicro Center staff can also assist you in selecting Agilent arrays but we are not able to assist in processing those microarrays.

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 eArray system (we can help you through the process of designing arrays). For anyone requiring microarray printing, we encourage you to contact Tom Volkert at the Whitehead Institute CGT.

QUALITY CONTROL

The BioMicro Center employs several quality controls as part of microarray analysis

RNA

All RNA samples are run on the Fragment Analyzer prior to labeling. The Fragment Analyzer results are used to determine the concentration of the RNA and the quality of the RNA based on their RNA Integrity Number (RIN) score.

Affymetrix Arrays

After scanning, 3' Affymetrix arrays are processed through a number of quality control steps to confirm the quality of the array. These steps create quality control plots that we distribute to the user.

QC PLOTS

In all cases, the plots are guidelines but not absolutes. Differences in the quality of starting RNA or source of the RNA can have a major impact on the plots. Biological replicates should behave similarly and we focus on changes where we find that is not true.

The RNA Degradation plot measure the relative intensity of probes at the 5' and 3' end of the average gene. Differences in the reverse transcription step can hive significant effects on the outcome. The precise slope of the line is not as important as having the lines have similar slopes.

Normalized Unscaled Standard Error (NUSE), provides a measure of relative chip quality derived from the residuals from the RMA model. NUSE scores above 1.05 or disconnected from the others are generally (though not always) considered poor.

The Affy QC pdf contains a number of metrics including box plots and histograms of intensity, QC scores for spike in controls and interarray correlation.

Fianlly, we use gcRMA for normalization. gcRMA normalized data is included in the data we distribute.

References

These plots are generated using a number of packages from bioconductor including:

affy:Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. 2004. affy---analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 3 (Feb. 2004), 307-315.
affyQCReport
affyPLM: Brettschneider J, Collin F, Bolstad BM, and Speed TP. (2007) Quality assessment for short oligonucleotide arrays. Technometrics.
LPE: Nitin Jain, Michael O'Connell and Jae K. Lee. Includes R source code contributed by HyungJun Cho <hcho@virginia.edu> (). LPE: Methods for analyzing microarray data using Local Pooled Error (LPE) method. R package version 1.20.0. http://www.r-project.org,

Bioconductor reference: Bioconductor: Open software development for computational biology and bioinformatics R. Gentleman, V. J. Carey, D. M. Bates, B.Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, and others 2004, Genome Biology, Vol. 5, R80

gcRMA: Jean(ZHIJIN) Wu and Rafael Irizarry with contributions from James MacDonald Jeff Gentry (). gcrma: Background Adjustment Using Sequence Information. R package version 2.18.1.

DATA ANALYSIS

Affymetrix Data Analysis

BioMicro Center uses Affymetrix Expression Console and GCRMA for analysis of Affymetrix data. Several files for each sample will be provided to the user:

.exp: Experiment information file
.dat: The image of the scanned probe array.
.cel: Cell Intensity File derived from .dat file.
.chp: Output file generated from the analysis of a probe array.
.rpt: Report File generated.
.txt: Text File is the same information as the .chp file in text format.

Additional data analysis help is available on a case by case basis. Please contact Stuart Levine if you need additional help with your analysis.

Agilent Data Analysis

Agilent Scanner is available to members of the MIT Community. We are currently running Feature extractor version 10.5. Please use iLabs to book time on the instrument.

LINKS

PRICES

Information for non-MIT labs

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.

@@ Line 2: / Line 2: @@
 [[Image:Presentation7.jpg|right|300px]]
-== Platforms ==
+The MIT BioMicro Center has extensive experience with several different types of microarray samples and platforms. Microarray labeling and hybridization are typically performed by BioMicro Center technicians, however our scanners, hybridization ovens and wash stations are available by request. NOTE: Assisted Agilent Microarray serves are no longer available. The Agilent oven and microarray scanner is available to users who prepare their own arrays.
-=== Affymetrix ===
-The MIT BioMicro Center preforms expression analysis using Affymetrix arrays. Our service includes:
-* Sample labeling and clean up using Affymentrix kits. ([http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf Product Manual])
-* Array hybridization
-* Data extraction and normalization
-We currently use arrays designed for whole transcript expression and 3’ expression analysis arrays for eukaryotic and prokaryotic samples.  We are exploring expanding our services to include SNP and Genotyping analyses. Please visit the [[BioMicroCenter:Ideas|IDEAS CENTER]] if you have input on the types of arrays you would like us to provide.
-=== Agilent ===
+== BioMicro Center Services ==
-The MIT BioMicro Center hosts a high-throughput Agilent Microarray Scanner, which has ability to measure fluorescence from two dyes simultaneously facilitates differential gene expression studies. We are currently planning to reintroduce Agilent array hybridization services in 2009.
+We are currently set up to primarily handle expression analysis. Our service includes:
-=== Nimblegen ===
+* Evaluation of sample quality using the [[BioMicroCenter:Advanced_analytical_Fragment_analyzer|Fragment Analyzer]]
-The BioMicro Center does not currently support the Nimblegen platform. We are currently discussing upgrading our Agilent Scanner to enable it to scan Nimblegen arrays as well. Anyone interested in using Nimblegen arrays should contact '''Stephen Bell'''.
+* Sample labeling and clean up
+* Array Hybridization
+* Data extraction and normalization
+We also have discounts with different vendors and can help you purchase your arrays.
+<br>
+=== SAMPLE TYPES ===
+The BioMicro Center has extensive experience handling samples for expression analysis.
+{|border=1
+ |-
+ ! Eukaryote
+ ! Min.RNA
+ ! Min.Vol.
+ ! Platforms
+ ! Notes
+ |-
+ | Prokaryote
+ | 1 μg
+ | 6 μL
+ | Affymetrix
+ | labeling using [http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=339 Ambion] for Affymetrix arrays
+ |-
+ | 3' Array (std)
+ | 50ng / ''100pg ''
+ | 6 μL
+ | Affymetrix
+ | labeling using Affymetrix reagents
+ |-
+ | Exon Array
+ | 50ng / ''100pg''
+ | 6 μL
+ | Affymetrix
+ | labeling using Affymetrix reagents
+|}
+<br>
-== Sample Submission Guidelines ==
+=== SAMPLE SUBMISSION ===
-We encourage researchers to contact us several days in advance of sample submission to allow us lead time to purchase any specialized Affymetrix arrays. While we keep a few arrays in stock, most arrays are ordered on an as needed basis. A list of some of the available arrays and their prices can be found [[Media:Affy_Arrays_081218.pdf| in this file]]. Information about Affymetrix arrays is available at http://www.affymetrix.com/products_services/index.affx
+We encourage researchers to contact us several days in advance of sample submission to allow us lead time to purchase any specialized arrays. All arrays are ordered on an as needed basis. A list of some of the available arrays and their prices can be found [[Media:Affy_Arrays_081218.pdf| in this file]]. Information about Affymetrix arrays is available at http://www.affymetrix.com/products_services/index.affx
 All submissions must be accompanied by a [[BioMicroCenter:Forms#Downloads | Affymetrix Submission Form]].
+<br>
+=== TURN AROUND ===
+The BioMicro Center will turn around your samples as quickly as possible. Currently the average turn around time is 1-2 weeks.
+<br>
-=== Affymetrix Sample Concentrations ===
+== ARRAY SELECTION ==
- '''ARRAY'''               '''Min.RNA'''   '''CONCENTRATION'''     '''NOTES'''
+Biomicro Center staff are available to assist you in selection of the proper microarray design. Newer Affymetrix microarrays take in to account ENCODE and RNAseq data to produce more efficient designs while older arrays have many probesets that are less useful. BioMicro Center staff can also assist you in selecting Agilent arrays but we are not able to assist in processing those microarrays.
- Prokaryote          10 ug     >0.6 ug/ul
- Euk. 3' Array        3 ug     >0.3 ug/ul
- [[3primeIVT|**New Kit**]]        0.1 ug                     http://www.affymetrix.com/products_services/reagents/specific/3_ivtexpresskit.affx
- Euk. Exon Array      2 ug     >0.5 ug/ul
+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].
-'''January 2009'''
+== QUALITY CONTROL ==
-** Affymetrix is retiring their old 3' Array kits in September. The [[3primeIVT|new kits]] are faster, require less starting material, and slightly less expensive (prices will be adjusted soon). Please indicate which type of kit you would like to use when submitting your sample.
+The BioMicro Center employs several quality controls as part of microarray analysis
+=== RNA ===
+All RNA samples are run on the [[BioMicroCenter:Advanced_analytical_Fragment_analyzer|Fragment Analyzer]] prior to labeling. The Fragment Analyzer results are used to determine the concentration of the RNA and the quality of the RNA based on their [[BioMicroCenter:RIN|RNA Integrity Number (RIN)]] score.
-Information about our evaluation of the new kits is [[3primeIVT|HERE]]
+=== Affymetrix Arrays ===
+After scanning, 3' Affymetrix arrays are processed through a number of quality control steps to confirm the quality of the array. These steps create quality control plots that we distribute to the user.
-=== Non-MIT Users ===
+==== QC PLOTS ====
-Please contact [[BioMicroCenter:People|Manlin Luo]] to arrange a drop off date and time. Samples on dry ice and chips at room temperature with a completed order form are shipped by overnight delivery to:
+In all cases, the plots are guidelines but not absolutes. Differences in the quality of starting RNA or source of the RNA can have a major impact on the plots. Biological replicates should behave similarly and we focus on changes where we find that is not true.
-   MIT BioMicro Center
-   Attention: Manlin Luo
-Ames Street, Building 68-316
-   Cambridge, MA 02139
-== Protocol ==
+The RNA Degradation plot measure the relative intensity of probes at the 5' and 3' end of the average gene. Differences in the reverse transcription step can hive significant effects on the outcome. The precise slope of the line is not as important as having the lines have similar slopes.
-<B><BIG>NEW!</BIG></B>
+Normalized Unscaled Standard Error (NUSE), provides a measure of relative chip quality derived from the residuals from the RMA model. NUSE scores above 1.05 or disconnected from the others are generally (though not always) considered poor.
-Affymetrix is switching their labeling kits to a new kit called [http://www.affymetrix.com/products_services/reagents/specific/3_ivtexpresskit.affx 3'IVT Express]<br>
-Information about the new kit can be found [[3primeIVT|HERE]]
+The Affy QC pdf contains a number of metrics including box plots and histograms of intensity, QC scores for spike in controls and interarray correlation.
+Fianlly, we use gcRMA for normalization. gcRMA normalized data is included in the data we distribute.
-== Analysis and Data Retrieval ==
+==== References ====
+These plots are generated using a number of packages from [http://www.bioconductor.org/ bioconductor] including:
+*affy:Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. 2004. affy---analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 3 (Feb. 2004), 307-315.
+*affyQCReport
+*affyPLM: Brettschneider J, Collin F, Bolstad BM, and Speed TP. (2007) Quality assessment for short oligonucleotide arrays. Technometrics.
+*LPE:   Nitin Jain, Michael O'Connell and Jae K. Lee. Includes R source code contributed by HyungJun Cho <hcho@virginia.edu> (). LPE: Methods for analyzing microarray data using Local Pooled Error (LPE) method. R package version 1.20.0. http://www.r-project.org,
+Bioconductor reference:   Bioconductor: Open software development for computational biology and bioinformatics R. Gentleman, V. J. Carey, D. M. Bates, B.Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, and others 2004, Genome Biology, Vol. 5, R80
+gcRMA:   Jean(ZHIJIN) Wu and Rafael Irizarry with contributions from James MacDonald Jeff Gentry (). gcrma: Background Adjustment Using Sequence Information. R package version 2.18.1.
+== DATA ANALYSIS ==
 === Affymetrix Data Analysis ===
-BioMicro Center will perform the initial data extraction of your samples using Affymetrix GCOS V.1.4. Affymetrix data is scaled to a average intensity of 500 per chip. Six files for each sample will be sent back to the user:
+BioMicro Center uses Affymetrix Expression Console and GCRMA for analysis of Affymetrix data. Several files for each sample will be provided to the user:
 * .exp: Experiment information file
@@ Line 68: / Line 106: @@
 Additional data analysis help is available on a case by case basis. Please contact [[BioMicroCenter:People|Stuart Levine]] if you need additional help with your analysis.
-=== Data Transfer ===
+=== Agilent Data Analysis ===
+Agilent Scanner is available to members of the MIT Community. We are currently running Feature extractor version 10.5. Please use [https://mit.ilabsolutions.com/sc/3381/ki-genomics-core-mit-biomicro-center/?tab=equipment iLabs] to book time on the instrument.
-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.
-The data will be placed on our server and can be downloaded by secure FTP or SSH.
-SSH can be downloaded from: ftp://metalab.unc.edu/pub/packages/security/ssh/SSHSecureShellClient-3.2.9.exe
-Your user name and password will be included in the email. To obtain your data, enter the following in your client:
-  Host:       bmc-150.mit.edu
-  user:       provided in email
-  Password:   provided in email
-Please contact [[BioMicroCenter:People|Stephen Goldman]] if you have difficulty obtaining your data.
-== Pricing ==
-Prices for sample processing do not include cost of arrays
-=== [[BioMicroCenter:MicroArrays|AFFYMETRIX ARRAYS]] ===
-Affymetrix processing prices '''do not''' include the cost of the array.  In order to be sure we have arrays on hand, we encourage users to contact us several days in advance of submitting samples.
-   '''ITEM'''                       '''[[CoreDeps|CORE LAB]]'''       '''MIT'''      '''NON-MIT'''     '''unit'''          '''NOTES'''
- Affy Expression Processing
-    Standard                    $260        $430       $645      per array    *kit will be retired Sep.2009
- Affy Exon Processing           $300        $535       $800      per array
- Common Affymetrix Arrays (in stock):
-    Human U133 Plus 2.0         $400
-    Human U133A 2.0             $250
-    Human Exon 1.0 ST           $400
-    Mouse 430 2.0               $400
-    Mouse 430A 2.0              $250
-    Mouse Exon 1.0 ST           $400
-    Yeast 2.0                   $175
- Full Price List:            [[Media:Affy_Arrays_081218.pdf| Affymetrix Prices]]
->> Price of 3' Expression arrays will be decreasing in early 2009 to reflect changes in the hybridization protocol.
-=== AGILENT SCANNER ===
-Agilent Scanner is available to members of the MIT Community without charge. If you are the first time user, contact [[BioMicroCenter:People|Stephen Goldman]] to set up a bionet account for your data storage. 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.
-=== [[2100BioAnalyzer|AGILENT BIOANALYZER]] ===
-   '''ITEM'''                       '''[[CoreDeps|CORE LAB]]'''       '''MIT'''      '''NON-MIT'''     '''unit'''          '''NOTES'''
-   RNA                           $10         $10        $15      per sample
-.
+== 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.