Microarray

The Research Core Facility is equipped with a state-of-the-artAffymetrix GeneChip Systemthat consists of the following components:

1. A GeneChip Hybridization Oven 645 for automated control of hybridization to the GeneChip arrays.
2. A GeneChip Fluidics Station 450for automated washing of chips and labeling of hybridized probes. This station can wash and stain four arrays simultaneously.
3. A GeneChip Scanner 3000 7G with Autoloader for obtaining high-resolution images of hybridization signals.
4. A GeneChip Workstation that controls the operation of the system, data collection and processing of initial raw data.
5. A bioinformatics system including Expression Console, Transcriptome Analysis Console, and Ingenuity Pathway Analysis.
    

Full service analysis includes Agilent RNA quality evaluation, sample labeling, hybridization, staining, scanning and data output.  In addition, the Core will accept labeled RNA samples for hybridization to and scanning of Affymetrix arrays.  The Core will not provide any reagents to investigators labeling their own samples.

Note:  The investigator is responsible for purchasing all expression arrays. Arrays can be directly shipped to the Core when ordered.

Affymetrix Array Prices (Inquire with ThermoFisher for current pricing)

For a complete list of Affymetrix arrays and part numbers, please visit ThermoFisher Scientific or contact our current Sales Representative, Lorenna Marques.  Lorenna can be reached by email at Lorena.marques@thermofisher.com.  To compare array types, please reference the data sheets above.

Clariom S vs Clariom D:  Clariom S arrays provide a snapshot view of the transcriptome, while Clariom D arrays provide a deeper view.  Clariom D arrays will allow you to interrogate > 500,000 transcripts, including splice variants, rare RNA isoforms and long-noncoding RNA.  Clariom S arrays will allow you to interrogate only well-annotated genes.

• Requirements may vary depending on the protocol being used. Please check with Core staff prior to sample preparation if you are unable to meet the minimum requirements. Special kits are available to process FFPE samples and samples with limiting quantities.
• Please fill in all information on the sample submission form.
• For full services, provide only sample (total RNA) and arrays.  All reagents are provided by the Core. Arrays can be directly shipped to the Core when ordered.
• Please do not submit your entire stock RNA sample to the Core. When eluting or re-suspending your RNA, make two aliquots for submission. This will prevent subjecting your stock RNA to unnecessary freeze-thaw cycles. Aliquot 3 ul for QC and 10 ul for processing.
• Use an established isolation method that produces intact RNA that is also free of interfering contaminants. It is best to elute in nuclease-free water. Sample purity is important, and this assay DOES NOT TOLERATE EDTA, salts, phenol, etc.
• In general, A260/A280 ratios should fall between 1.8-2.1; however, do not discard samples with ratios that fall outside of this range. These ratios may vary depending on tissue type, buffer content, etc.
• An Agilent analysis will be run to assess the integrity of your total RNA prior to processing. In general, the RNA Integrity Number (RIN) generated from the Agilent analysis should be at least 8.0.
• DNase digestion is required when using whole transcriptome arrays (HTA, MTA, Clariom). DNase digestion is not required when using 3' arrays. Please ask Core staff if you need clarification. If you must DNase-treat your samples, make sure you use a good quality DNase that will not degrade your RNA and that you deactivate the DNase appropriately.
• Replicates (at least n=3) are recommended for proper normalization and differential analysis.
• Please refer to http://www.ncbi.nlm.nih.gov/geo/info/MIAME.html for more information relating to microarray standards and data deposition into public repositories. The MIAME (Minimum Information About A Microarray Experiment) guidelines outline the minimum information that should be included when describing a microarray experiment.  Many journals and funding agencies require microarray data to be MIAME compliant. The GEO (Gene Expression Omnibus) repository submission procedures are designed to follow the MIAME checklist.
• Samples are processed according to the order in which they are received. Turnaround time is typically 7 to 14 days from receipt of samples.
• If processing samples outside of LSU Health, send total RNA overnight on dry ice to the address below. Do not ship samples for delivery on a weekend. Samples must be received Monday through Friday. Notify Core staff prior to shipping to ensure we will be available to receive your samples. Samples may be shipped to:

Camille Abshire
LSU Health Shreveport
Research Core Facility, BRI F6-15
1501 Kings Hwy
Shreveport, LA 71103

Notes:  

1. Samples that fail quality control measures using the Agilent TapeStation or the Qubit analyzer will be billed for the cost of the reagents associated with these analyses. In addition, samples that fail labeling for Affymetrix microarray will be billed for the cost of the reagents used in these procedures, if the failure is due to sample quality issues. These measures are necessary, as the RCF needs to recoup the associated consumable costs for these procedures.
2. Samples will be put in the queue for processing ONLY after they have been received and pass all quality control measures.

Transcriptome Analysis Console, for gene expression analysis of Affymetrix microarrays, is available for free download from the ThermoFisher website:

Ingenuity Pathway Analysis (IPA) is available through the Core to LSU System investigators only.  Investigators must purchase an individual account on our license; this is the only option, as Qiagen changed the license terms.  Qiagen will directly invoice you, but you must contact Core staff prior to purchase.  The cost varies from year to year but usually averages around $3,500 (please inquire with Core staff for assistance obtaining a quote).  

Routine consultations with Core staff are free; however, extensive consultation for technical and bioinformatics support will carry a consultation fee of $50/hour. 

Transcriptome Analysis Console (TAC) is available as a free download from ThermoFisher.  It is only compatible with a Windows operating system and requires a minimum of 8GB of RAM.  TAC is installed in the RCF Computer Lab.
The following data files will be delivered to the investigator:

• Experiment File *.ARR: This file contains the parameters of the experiment such as probe array type, experiment name, equipment parameters and sample description. This file is not used for analysis, but may be needed to open other files for the designated chip experiment.
• Image Data File *.DAT: This is an image file generated after scanning the array. This file can be opened in some viewer packages to assess the quality of scanning. It is used in Expression Console to generate the *.CEL file.
• Image File *.JPG: This is a snapshot of the .DAT file.
• Cell Intensity File *.CEL:  This binary file is the result of low level analysis performed from the *.DAT image file. It is can be used as for further analysis.
• Probe Array Results File *.CHP: This is a gene level summarization of the CEL file using the Affymetrix’ normalization algorithms. It is exported from Expression Console. It can also be used as the base file for further analysis; however, there are many other algorithms that have been adopted by the scientific community other than those provided by Affymetrix software, hence the reason why CEL files are often preferred over CHP files for analysis.
• Report File *.RPT: The report file is generated from the CHP file. This expression report summarizes information about expression analysis settings and probe set hybridization intensity data.
• Audit File *.ADUIT: The audit file is an XML file that tracks the processing of each physical array processed by AGCC. An audit file is produced for each physical array and tracks all the processing steps that were performed on the array, including multiple scans and regridding.

In addition to the files above, intensity values will be provided in an Excel spreadsheet. Upon request, the CAIPP Bioinformatics and Modeling Core can provide help with specific needs.

CAIPP Bioinformatics and Modeling Core Team:

Dr. Rona Scott
Director of CAIPP Bioinformatics and Modeling Core
Rona.Scott@lsuhs.edu

Jian Wang, PhD
Assistant Director of CAIPP Bioinformatics and Modeling Core
Jian.Wang@lsuhs.edu