Iowa Institute of Human Genetics

  • Microarray

    Sample Submission

    Total RNA for Expression Profiling Studies

    1. Sample requirements:

    a. Minimum of 1 ug total RNA. 2 ug is preferable.
    b. Minimum of 10 ul volume-suspended in nuclease-free water.
    c. All samples normalized to 100 ng/ul. Concentration is just as important as the total quantity.
    d. If the above sample quantity requirements are not possible, please contact us to explore whether other options may be available for your samples. Alternatives exist.

    2. Isolation of total RNA with Qiagen RNeasy is the preferred method (other equivalent methods are acceptable). If Trizol is used, a subsequent purification with RNeasy is recommended to remove all traces of residual organic carryover.

    3. Do not use 'inert' carriers (e.g., glycogen, tRNA, etc.) in the RNA isolation and purification.

    4. DNAse treatments are strongly encouraged and not considered 'optional'.

    5. If elution buffer is NOT nuclease free H2O, please provide a 50 ul aliquot of the elution solution used for proper blanking of the spectrophotometer during our quality control assessments.

    6. Before submitting your samples, please complete the online Microarray Request Form. Bring a printed copy of your request with your samples.

    7. Please give advance notice of submission date and time.

    8. Samples are not officially in our queue until all samples are received and the Quality Control (QC) analysis is completed. At that point, the appropriate Illumina Beadchips or Affymetrix GeneChip arrays and their reagents will be ordered and the project becomes part of the processing queue. We do not order reagents or arrays in advance of sample reception and QC analysis. Projects are processed in the order they are received according to the requirements listed above.

    DNA for Array-based Genotyping and Mapping

    Please consult with the IIHG Genomics Division microarray personnel before starting a SNP Genotyping or Mapping Array project. It is our experience that the most important factor in the success of mapping or genotyping arrays is the quality and correct quantity of the DNA samples.

    Below are our guidelines for providing samples that typically give successful array data.

    1. Suspend DNA samples in either water, Tris (Qiagen EB is fine) or low TE (0.1 mM EDTA). Higher concentrations of EDTA (as in TE) could be inhibitory to some enzymatic steps in the process.

    2. Mix the samples WELL (It is ok to vortex for the purpose of the mapping arrays. Do not vortex if the same sample will be used for other applications).

    3. Quantify samples in duplicate using a spectrophotometer (e.g. Nanodrop) or fluorometer (e.g. Qubit). All samples should be in the range of 50-100 ng/ul.

    • Samples under 50 ng/ul need to be concentrated.
    • Please dilute samples with DNAse free water if the concentration is above 100 ng/ul. (We recommend aiming for 80 ng/ul).
    • The IIHG Genomics Division has a NanoDrop spectrophotometer available for use to measure concentrations. We will guide you on how to use it. Print out both the results table and the profiles of the samples.

    4. Run all samples on a ~1% agarose gel along with a positive control of 50 ng/ul genomic DNA that we will provide and a 1 kb-plus ladder. Load 3 ul of sample and 6 ul of ladder. You want to see a high molecular weight band of approximately the same brightness for all the samples.

    5. Please talk to us about sample volume and arrangement on the submission plate. We usually request 20 ul of each sample, however, there are exceptions. Also, the various microarray sample prep workflows start with different configurations of the samples on the plate.

    6. Please provide the Nanodrop results (table and profiles) and the gel image along with your samples.