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Genomics Core Laboratory
Affymetrix™ GeneChip™

The Genomics Core Laboratory houses a complete Affymetrix™ GeneChip™ system including a hybridization oven, an automated fluidics station allowing parallel processing of 4 chips and a high resolution scanner. Also available is the Affymetrix™ Total Desktop Mining Solution suite of software for array reading, analysis and data mining.

Services Offered

Full Chip Service: Bring your total RNA (10ug total which has been Trizol isolated and then RNAeasy (Qiagen) purified. The facility then performs quality analysis of total RNA, cRNA synthesis, testing of labeling, hybridization, scanning, and data extraction.

Hybridization and Scanning Only: Bring your cRNA probe labeled with biotin to the facility. The facility will add the proper control oligos and cRNA, hybridize, wash, scan and extract raw data. The users assume all responsibility for failed hybridizations. Turn around time is usually 1 week. As soon as the results are ready, the investigator will be contacted via email.

Sample Submission Form

GeneChip™ Expression Analysis Experiments
GeneChip™ Expression analysis experiments should include the following major steps:

1. Experimental Design
2. RNA isolation
3. Target cRNA preparation
4. Hybridization to the test array
5. Test array data analysis
6. Hybridization to the standard array

1. Experimental Design: It is absolutely essential to plan and execute the experiments with utmost care. The experimental model/system should be well characterized or well defined with an independent experimental verification.  It is recommended that all experimental treatments be carried out in triplicates to compensate for biological and experimental variation. In vitro experiments using cultured cells should be conducted three different times (not three replicates performed on the same day) strictly following the same experimental procedures. Tumor specimens should be devoid of adjacent tissues and if possible, should be micro dissected to obtain as pure a tumor sample as possible. Cell populations may also be further purified using cell-sorting techniques such as FACS. Dead cells also should be removed by density centrifugation. For comparative gene expression analysis, it is essential that all the experimental conditions such as temperature, CO₂, media, reagents, and sample processing be kept identical for all samples.

2. RNA isolation: The quality of the RNA is the single most important determinant of success of a GeneChip™ analysis assay. Particularly, differential degradation of RNA can lead to erroneous conclusions about both the relative and absolute mRNA levels in the specimens. Although either mRNA or total RNA can be used as starting material, we prefer total RNA for two reasons: (1) isolating total RNA is easier and more economical than isolating mRNA, and (2) there is loss of starting material during mRNA purification and consequently, more mRNA is required to achieve sensitivity similar to that of the total RNA. In addition there may also be differential loss of individual mRNAs.

We recommend TRIzol reagent for isolation of total RNA from tissue specimens as well as cultured and blood cells. Total RNA isolated using TRIzol should be further purified using the Qiagen RNeasy cleanup procedure.

The A260/A280 ratio should be at least 1.9 for pure RNA. The quality of RNA should also be assessed by agarose gel electrophoresis. The agarose gel profile should exhibit a 28S band that is 2 times more intense than 18S ribosomal RNA. It is important that the total RNA is free from genomic DNA contamination. There are precautions to be taken while using the RNeasy kit to avoid genomic DNA contamination.  If genomic DNA contamination is present, it is essential to remove it by DNase treatment. DNase then can be removed by heat inactivation followed by RNeasy cleanup using Qiagen kit.

The minimum amount of total RNA required for GeneChip™ analysis is 10 μg.

3. Target (labeled cRNA) Preparation: Good quality total RNA is used as starting material to obtain labeled cRNA. In the first step, single stranded cDNA is synthesized by reverse transcription using poly-A RNA present in the starting total RNA sample. Single stranded cDNA is then converted into double stranded cDNA which is extracted with phenol/chloroform and then precipitated with ethanol. An in vitro transcription (IVT) reaction is then carried out in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA from the double stranded cDNA. The resulting cRNA is then fragmented in the presence of heat and Mg⁺⁺, before hybridization to the test arrays.

4. Test array hybridization: Test arrays are relatively inexpensive and are used as an assessment tool for determining target quality and labeling efficiency. Fragmented cRNA is hybridized to the test array for 16 hours at 45⁰ C. The test array is then washed and stained with streptavidin-phycoerythrin using the fluidics station and then scanned using gene array scanner.

5. Hybridization to the Standard array: If the data obtained from the test array is satisfactory, the sample is hybridized to the standard array for 16 hours at 45⁰ C. The standard array is then washed and stained using the fluidics station and then scanned. The images will be analyzed using Micro array Suite Software and comparison analysis will be carried out according to the instructions provided by Affymetrix™.
 

The preceding information is credited to the following website:
The Affymetrix GeneChip Resource at Yale,
http://info.med.yale.edu/wmkeck/affymetrix/analysis.htm
and is used with permission.

Research  |  Medical College of Georgia

Please email comments, suggestions or questions to:
Abhijit Afzalpurkar, aafzalpurkar@mcg.edu
December 05, 2006