bioinformatics banner
slider

Agilent 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 Agilent microarray platform. These services include evaluation of nucleic acid quality, labeling of samples with modified nucleotides (cy dyes), microarray hybridization, microarray scanning, feature extraction and annotation of scanned images. Agilent microarrays that are supported in the core facility offer a diverse set of tools for investigators that includes measuring gene and miRNA expression levels, DNA copy number profiling, location analysis of DNA binding proteins, and defining DNA methylation status. In addition to catalog microarrays that are available for these applications, the Agilent platform offers significant flexibility in the design and printing of customized microarrays.

Agilent 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, however it is not included with the RNeasy kit and must be bought separately.
  • Do not overload Qiagen columns during DNA purification. Overloading columns can introduce impurities (guanidine, protein, carbohydrate) that can have inhibitory activity during the 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.
  • A quantity of 25-1000 ng of total RNA is recommended for cost-effective labeling of samples in preparation for Agilent gene expression microarray analysis. The sample should be provided to the Microarray Core Facility in a volume of 10 ul at a concentration between 5 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.
  • Most catalog Agilent gene expression microarrays are printed in an 4-plex format. Therefore, when developing the experimental design, one should plan on using a multiple of four microarrays.
  • Pricing for Agilent Gene Expression Microarray Services

Agilent miRNA Expression Microarrays

  • RNA should be provided as total RNA. Preferred sample preparation methods include the Qiagen miRNeasy kit.
  • Do not use the standard Qiagen RNeasy kit. This kit will result in substantial loss of RNA molecules less than 200 nucleotides in length which includes small RNA.
  • Do not overload Qiagen columns during DNA purification. Overloading columns can introduce impurities (guanidine, protein, carbohydrate) that can have inhibitory activity during the labeling protocol or introduce background noise during the hybridization process.
  • RNA samples should be treated with DNase. This step should be included and performed during the Qiagen miRNeasy purification methods. DNase for this procedure should be acquired from Qiagen.
  • 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 miRNeasy spin column. Residual DNA can also be removed during this cleanup step by including DNase.
  • A quantity of 100 ng of total RNA is required for the small RNA labeling reaction. We request additional material so that we can perform quality control steps including measurement of the concentration on a NanoDrop and qualitative analysis on a bioanalyzer. The sample should be provided to the Microarray Core Facility in a volume of 6 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 small RNA labeling if concerns arise from this assay about the quality of any sample within the submitted request.
  • Agilent miRNA microarrays are printed in an 8-plex format. Therefore, when developing the experimental design, one should plan on using a multiple of 8 microarrays.
  • Pricing for Agilent miRNA Expression Microarray Services

Agilent Comparative Genomic Hybridization (CGH) 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 labeling 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.
  • The recommended quantity of genomic DNA needed for hybridization on a 1M or 244K microarray is 1.5-3.0 μg. The recommended quantity of genomic DNA needed for hybridization on a 105K or 44K microarray is 0.5-1.5 μg.
  • A volume of 25 μl of genomic DNA sample adjusted to a concentration of 75-150 ng/μl is required for either 1M or 244K CGH microarrays. A volume of 25 μl of genomic DNA sample adjusted to a concentration of 50-75 ng/μl is required for either 105K or 44K CGH microarrays. It is preferred that you provide a quantity on the high end of these concentration ranges.
  • 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. Samples that contain low molecular weight DNA will not perform well on Agilent microarrays.
  • Pricing for Agilent Comparative Genomic Hybridization Microarray Services

Agilent ChIP-on-chip Microarrays

  • Genomic DNA should be fragmented to a size range of 200-600 bp prior to immunoprecipitation. Preferred methods of fragmentation include sonication via a Diagenode Bioruptor, enzymatic digestion using an Active Motif ChIP-IT kit (http://www.activemotif.com/catalog/9.html), or using the Covaris Adaptive Focussed Acoustics technology. We discourage the use of probe-based sonicators which can introduce significant sample to sample variability due to inconsistencies in the size distributions of the fragmented samples.
  • The size distribution of a matched input DNA for each sample can be verified by running an aliquot of the sample on an Agilent High Sensitivity DNA Bioanalyzer chip (1 ng) or on a 2% agarose gel (50-100 ng). The DNA should show uniform size distribution in the 200-600 bp range. The presence of a substantial fraction of higher molecular weight DNA (>1 kb) indicates incomplete fragmentation which could introduce amount of amplified ChIP or input DNA for a ChIP-on-chip labeling reaction is 2-3 μg of DNA. The recommended kit for amplification of ChIP-DNA is the Sigma Genomeplex Whole Genome Amplification kit.
  • We discourage the use of probe-based sonicators as these routinely introduce significant sample to sample variability due to excessive heating and denaturation of samples in small volumes and irregular size distributions between samples that are sonnicated.
  • Gloves and filter tips should be used during all stages of handling ChIP-DNA and Input DNA. The yield of DNA from an immunoprecipitation reaction is very small. 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 a significant contribution to the final amplified ChIP product.
  • The amplified ChIP DNA samples should be provided to the Microarray Core Facility in a volume of 30 μl. The Microarray Core Facility can assist concentrating samples that are provided in volumes up to 100 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.
  • Avoid organic extraction methods (such as phenol or Trizol) to purify genomic DNA and ChIP-DNA samples. Organic carryover can inhibit the enzymatic reactions used in microarray labeling reactions. If an organic extraction method is used, this should be followed by purification on a Qiagen spin column. Preferred sample purification kits include the Qiagen PCR purification kit.
  • Carrier DNA such as salmon sperm DNA, calf thymus DNA or other DNA based carriers that are used as a blocking agent at any step during the immunoprecipitation process can adversely affect the microarray experiment. Carrier DNA that is added to your sample will also be present in the final amplified ChIP-DNA product provided to the core facility. This could result in over-estimating the quantity of ChIP-DNA derived from your sample and result in reduced hybridization signal on the microarray.
  • Magnetic Beads (Dynabeads) do not readily absorb random DNA from the immunoprecipitation cocktail and therefore are preferred over Sepharose, Sephadex etc.
  • ChIP DNA sample concentration is very low and reliable readings cannot be obtained using a spectrophotometer. Accurate concentration readings can be obtained for these samples using a PicoGreen assay such as that performed on an Invitrogen Qubit.
  • Pricing for Agilent ChIP-on-chip Microarray Services

Agilent HybMap Expression Microarrays

  • HybMap is a novel RNA-DNA hybridization mapping technique that uses an antibody directed against an RNA-DNA hybrid to detect RNA molecules that are hybridized to a high-density DNA oligonucleotide tiling array.
  • The HybMap technique is used with custom designed Agilent microarrays.
  • RNA should be provided as total RNA. Preferred sample preparation methods include the Qiagen RNeasy 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 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.
  • A quantity of 1-5 μg of total RNA is recommended for HybMap expression microarray analysis. The sample should be provided to the Microarray Core Facility in a volume of 10-20 ul at a concentration between 50 ng/μl and 500 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 Agilent HybMap Expression Microarray Services