Affymetrix GeneChip® - Global Expression Profiling

Service option 1: Total RNA for 3' Expression Analysis

Total RNA sample requirements for 3' expression profiling using Affymetrix GeneChip arrays are 10μg of total RNA at a concentration of at least 1 ug/ul. While the lab will perform QC analysis of all sample submitted for microarray analysis, it is advisable that the investigator perform a preliminary assessment of sample quality before sending samples to the lab. Failure to perform standard QC analysis of your RNA samples will result in sample processing delays.

All samples, genome GeneChips, and a fully completed sample submission form should be delivered to the DNA Array Lab, located in the Thomas Building, DE-740.

NOTE: If you are starting from whole blood samples please contact Alyssa Dawson 206.667.5614, adawson@fhcrc.org) before submitting the RNA.

Affymetrix request forms:
If you are using a FHCRC project id for billing, please fill out the Internal submission form.

If billing is to be submitted to the University of Washington or other external institution, fill out the External submission form.

Service option 1: Sample QC

Quality control of total RNA includes:

QC test for RNA degradation. The resource has two Agilent 2100 Bioanalyzers that are used to assess the initial RNA quality. To learn more about how to interpret Bioanalyzer results, please visit the Agilent web site.

QC test for protein and organic contaminates. The resource has a NanoDrop ND-1000 spectrophotometer that is used to assess sample purity. Employing a UV scan from 210nm - 310 nm, the following criteria must be met:

• 1.8 ≤ A₂₆₀/ A₂₈₀ ≤ 2.1
• A₂₆₀ / A₂₃₀ ≥ 1.7
• A₃₁₀ ~ 0

To ensure that the above criteria are based on sufficient absorption measurements, we also require an A₂₆₀ > 0.3 when performing this analysis.

In instances where one or more samples do not pass our initial QC tests, we will not proceed with the project and immediately contact the submitter with our findings.

Service option 1: Preparation of fragmented cRNA target

The Genomics Resource staff generates biotin-labeled cRNA targets for hybridization with strict adherence to the sample preparation protocols outlined in the Affymetrix GeneChip Expression Analysis Technical Manual. Samples will be closely monitored throughout this process using the Agilent 2100 Bioanalyzer and the NanoDrop ND-1000 spectrophotometer.

Service option 1: Run Test Hybridization

The Affymetrix GeneChip Test3 Array (P/N900342; supplied by the resource) contains a small subset of housekeeping genes from various organisms and is used by the Genomics Resource staff to assess target quality and labeling efficiency prior to utilizing expression profiling GeneChip products.

To learn more about how to interpret Test3 Array results, please refer to the following documents.

Test hybridization report
Data Analysis Manual

In instances where one or more samples do not pass our Test3 Array QC tests, we will not proceed with the project and immediately contact the submitter with our findings.

Service option 2: Total RNA Preparation

Total RNA sample requirements for 3'expression profiling using Affymetrix GeneChip arrays are 10μg of total RNA at a concentration of at least 1μg/ul.

Service option 2: Quality Control

Quality control of total RNA includes QC test for RNA degradation and QC test for protein and organic contaminates. The following criteria must be met:

• A₂₆₀ ≤ 1.8 / A₂₈₀ ≤ 2.1
• A₂₆₀ / A₂₃₀ ≥ 1.7
• A₃₁₀ ~ 0

Service option 2: Labeled cRNA for 3' Expression Analysis

Labeled cRNA sample requirements for expression profiling using Affymetrix GeneChip arrays are 15ug of fragmented biotin-labeled cRNA in a total volume of 40ul submitted in a 1.5ml tube. For labeling protocols please refer to the Affymetrix Expression Analysis Technical Manual. The Genomics Resource lab will not perform QC analysis of fragmented biotin-labeled cRNA samples submitted for microarray analysis, so it is advisable that the investigator perform assessment of sample quality before sending samples to the lab.

All samples, GeneChips, and a fully completed sample submission form should be delivered to the DNA Array Lab, located in the Thomas Building, DE-740.

NOTE: If you are starting from whole blood samples please contact Alyssa Dawson 206.667.5614, adawson@fhcrc.org) before submitting the RNA.

Affymetrix request forms:
If you are using a FHCRC project id for billing, please fill out the Internal submission form.

If billing is to be submitted to the University of Washington or other external institution, fill out the External submission form.

Service option 2: OPTIONAL: Run Test Hybridization

The Genomics Resource lab will not perform QC analysis of fragmented biotin-labeled DNA samples submitted for microarray analysis, so it is advisable that the investigator performs an assessment of sample quality before sending samples to the lab.

The Affymetrix GeneChip Test3 Array (P/N900342; stocked by the resource) contains a small subset of housekeeping genes from various organisms and is used by the Genomics Resource staff to assess target quality and labeling efficiency prior to utilizing expression GeneChip products.

To learn more about how to interpret Test3 Array results, please refer to the following documents.

Test hybridization report
Data Analysis Manual

Continue to next steps in the process.