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Affymetrix Microarrays

Contents

Introduction

The Microarray Core Facility is operated as a full service facility and is therefore able to provide experimental handling and support for all aspects of the Affymetrix microarray platform. These services include evaluation of nucleic acid quality, labeling of samples with modified nucleotides (biotin), microarray hybridization, microarray scanning, feature extraction and annotation of scanned images. Affymetrix microarrays that are supported in the core facility offer a set of tools for investigators that includes the measurement of gene expression and and miRNA expression levels, SNP profiling and DNA copy number profiling.

Affymetrix Gene Expression Microarrays

  • RNA should be provided as total RNA. Preferred sample preparation methods include the Qiagen RNeasy kit. If working with plant or fungi, it is recommended to use the Qiagen RNeasy Plant mini kit.
  • RNA samples should be treated with DNase. This step should be included and performed during the Qiagen RNeasy purification methods. DNase for this procedure should be acquired from Qiagen.
  • Do not overload Qiagen columns during DNA purification. Overloading columns can introduce impurities (guanidine, protein, carbohydrate) that can have inhibitory activity during the Affymetrix SNP labeling protocol or introduce background noise during the hybridization process.
  • Avoid organic extraction methods (such as phenol or Trizol) to purify total RNA. Organic carryover can inhibit the enzymatic reactions that take place during labeling of the sample which can result in lower signal on the hybridized microarray. If an organic extraction method is used, it is recommended to follow the initial organic extraction with cleanup on a Qiagen RNeasy spin column. Residual DNA can also be removed during this cleanup step by including DNase.
  • Total RNA should be provided to the Microarray Core Facility in a volume of 15 ul at a concentration between 50 ng/μl and 100 ng/μl.
  • Do not send a sample volume/quantity in excess of the guidelines above. We are unable to provide a long term storage solution for excess samples. The unused portion of these samples is discarded after the experiment has been successfully completed.
  • Total RNA quality will be validated by running an aliquot of the sample on an Agilent Bioanalyzer RNA NanoChip. The client will be contacted following the Bioanalyzer run and prior to gene expression labeling if concerns arise from this assay about the quality of any sample within the submitted request.
  • Pricing for Affymetrix Gene Expression Microarray Services

Affymetrix 250K SNP MIcroarrays

  • Genomic DNA should be provided as high molecular weight DNA. Preferred sample preparation methods include the Qiagen DNeasy kit and the Qiagen Genomic-Tip System.
  • DNA samples should be treated with RNase. This step should be included and performed during the Qiagen DNeasy or Qiagen Genomic-tip purification methods.
  • Do not overload Qiagen columns during DNA purification. Overloading columns can introduce impurities (guanidine, protein, carbohydrate) that can have inhibitory activity during the Affymetrix SNP labeling protocol or introduce background noise during the hybridization process.
  • Avoid organic extraction methods (such as phenol or Trizol) to purify genomic DNA. Organic carryover can inhibit the enzymatic reactions used in Illumina library preparation. If an organic extraction method is used, this should be followed by purification on a Qiagen spin column. To remove residual organic contamination in a DNA sample it is recommended to use the Qiagen DNeasy kit.
  • Gloves and filter tips should be used during all stages of handling genomic DNA for SNP microarrays. The failure to use gloves or filter tips can contaminate the sample with human DNA from skin or by DNA that is introduced through the aerosol of pipettes. These sources of DNA can introduce background hybridizataion to your hybridized SNP microarray.
  • A volume of 10 μl of high molecular weight genomic DNA that has been adjusted to a concentration between 50-100 ng/μl should be delivered to the Microarray Core Facility for Affymetrix 250K SNP microarray assays. Concentrations below this range will have insufficient DNA for performing the Affymetrix SNP 5.0 or Affymetrix SNP 6.0 assay.
  • Do not send a sample volume/quantity in excess of the guidelines above. We are unable to provide a long term storage solution for excess samples. The unused portion of these samples is discarded after the experiment has been successfully completed.
  • Genomic DNA quality can be accessed by running approximately 50 ng of the sample on a 1% agarose gel stained with Ethidium Bromide. Intact genomic DNA should appear as a high molecular weight (>10,000 bp) band with no lower molecular weight smear. A small amount of low molecular weight smear may be acceptable; however, this should be limited. A significant amount of visible low molecular smearing may be detrimental to library generation. Low molecular weight DNA samples will not perform well on Affymetrix microarrays.
  • Pricing for Affymetrix 250K SNP Microarray Services

Affymetrix SNP 5.0/6.0 Microarrays

  • Genomic DNA should be provided as high molecular weight DNA. Preferred sample preparation methods include the Qiagen DNeasy kit and the Qiagen Genomic-Tip System.
  • DNA samples should be treated with RNase. This step should be included and performed during the Qiagen DNeasy or Qiagen Genomic-tip purification methods.
  • Do not overload Qiagen columns during DNA purification. Overloading columns can introduce impurities (guanidine, protein, carbohydrate) that can have inhibitory activity during the Affymetrix SNP labeling protocol or introduce background noise during the hybridization process.
  • Avoid organic extraction methods (such as phenol or Trizol) to purify genomic DNA. Organic carryover can inhibit the enzymatic reactions used in Illumina library preparation. If an organic extraction method is used, this should be followed by purification on a Qiagen spin column. To remove residual organic contamination in a DNA sample it is recommended to use the Qiagen DNeasy kit.
  • Gloves and filter tips should be used during all stages of handling genomic DNA for SNP microarrays. The failure to use gloves or filter tips can contaminate the sample with human DNA from skin or by DNA that is introduced through the aerosol of pipettes. These sources of DNA can introduce background hybridizataion to your hybridized SNP microarray.
  • A volume of 15 μl of high molecular weight genomic DNA that has been adjusted to a concentration between 50-100 ng/μl should be delivered to the Microarray Core Facility for either the Affymetrix SNP 5.0 or the Affymetrix SNP 6.0 microarray assay. Concentrations below this range will have insufficient DNA for performing the Affymetrix SNP 5.0 or Affymetrix SNP 6.0 assay.
  • Do not send a sample volume/quantity in excess of the guidelines above. We are unable to provide a long term storage solution for excess samples. The unused portion of these samples is discarded after the experiment has been successfully completed.
  • Genomic DNA quality can be accessed by running approximately 50 ng of the sample on a 1% agarose gel stained with Ethidium Bromide. Intact genomic DNA should appear as a high molecular weight (>10,000 bp) band with no lower molecular weight smear. A small amount of low molecular weight smear may be acceptable; however, this should be limited. A significant amount of visible low molecular smearing may be detrimental to library generation. Low molecular weight DNA samples will not perform well on Affymetrix microarrays.
  • Pricing for Affymetrix SNP 5.0/6.0 Microarray Services