BioMicroCenter:Microarrays

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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.

Contents

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.

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