3' Gene Expression Arrays

  • The Microarray Genomics Core Facility offers a gene expression profiling service that uses oligonucleotide microarrays. By using this method, the RNA of interest is converted into cDNA, then amplified and labeled with biotinylated nucleotides by in vitro transcription to produce biotinylated cRNA. The cRNA is hybridized to a microarray chip. The bound cRNA is stained with fluorescent streptavidin. The microarray chip is then scanned using a laser, and the positions and intensities of the fluorescent emissions are captured. These measurements provide the basis of subsequent biostatistical analysis. Experimental turn-around times are usually three business days.

    Isolation
    The analysis begins with total RNA or mRNA that is provided by an investigator. In our practice, isolating total RNA from mammalian tissue using a reagent such as TRIzol, followed by additional purification using RNeasy from QIAGEN, yields RNA that produces satisfactory microarray data. In addition, successful isolation of total RNA from mammalian cells has been purified and eluted using RNeasy. Good quality mRNA has been successfully isolated from mammalian cells using QIAGEN’s Oligotex Direct mRNA kit and from total RNA using the Oligotex mRNA kit. If mammalian tissue is the source of mRNA, total RNA should be first purified using a reagent such as TRIzol, and then using QIAGEN’s mRNA isolation procedures or other commercial kits. Modifications of these methods, as well as other methods, are also known to produce suitable total RNA. Please contact our core facility if you are uncertain as to whether an RNA extraction method that you are considering is suitable.

    Quantity
    The Microarray Genomics Core prefers to start the GeneChip® Expression Arrays with total RNA samples concentrated at a minimum of 500ng/µL. Investigators need to provide documentation of sample quantity before experiments begin. All sample quantities are rechecked on the NanoDrop 1000 Spectrophotometer. Once the quantity has been rechecked and verified for each sample, your experiment will begin. If quantity is an issue for any sample, then the investigator will be notified immediately to discuss the next course of action. We strongly encourage investigators to submit a minimum of 5µg of total RNA in 10µL or 1µg of mRNA in 2µL for experimental purposes when using the One-Cycle Affymetrix GeneChip® Amplification kit. We have found through experience that a greater amount of total starting RNA produces the best results. However, we realize that this is not always a feasible option so you can provide anywhere from 1 to 15µg of total RNA or 0.2 to 2µg of mRNA per sample for an experiment, but keep in mind that your samples may not be as robust below our recommendation. Please provide an extra 2ug of total RNA or mRNA in addition to the experimental quantity you choose to submit in order to account for the protocol checkpoints and any concentration variations, should your sample concentrations be off.

    When using the Two-Cycle Affymetrix GeneChip® Amplification kit, we recommend that investigators submit 100ng of total RNA in 2µL. If this is not possible, you can provide anywhere from 10 to 100ng of total RNA for an experiment, but again keep in mind that your samples may not be as robust below our recommendation. Please provide an extra 200ng of total RNA in addition to the experimental quantity you choose to submit in order to account for the protocol checkpoints and any concentration variations, should your sample concentrations be off.

    Quality
    The quality of the RNA is essential to the overall success of a gene expression array. We require an agarose gel picture of the submitted samples that clearly defines the prominent 28S and 18S rRNA bands for each sample. Investigators have to provide this documentation of sample quality before experiments begin. The spectrophotometric absorbances from the NanoDrop 1000 are checked at A260, A280 and A230 nm for each sample. The A260/280 ratios for each sample should be close to 2.0 for pure RNA samples (ratios between 1.7 and 2.1 are acceptable), while the A260/230 ratios for each sample should be between 1.8 and 2.2 for high-quality RNA. In addition, all sample qualities are rechecked on the Agilent 2100 Bioanalyzer. Once the quality has been verified for each sample, your experiment will begin. If quality is an issue for any sample, then the investigator will be notified immediately to discuss the next course of action.