Pathway-Centric Tools and Technology™

Ten Most Frequently Asked Questions about the GEArray Microarray

1. What is unique about the GEArray?

GEArray® (Gene Expression Array) Focused DNA Microarrays are the ideal tools for gene expression studies. They allow you to profile the expression of multiple genes on a single array using a simple hybridization procedure and common laboratory equipment. Instead of including a large number of unrelated genes in a single array, each GEArray includes a thoroughly researched set of relevant, pathway- or disease-focused collection of genes. This unique, knowledge-based design incorporates genes involved in a specific biological pathway, genes related to a particular disease state, or genes with similar functions or structural features. With the combination of the high binding capacity of nylon matrix, cutting-edge non-contact array printing technology, advanced array labeling technology and chemiluminescence detection, the GEArray system delivers superior sensitivity, reliability, reproducibility and robust performance with as little as 1 microgram of total RNA. Because of their focused design, data handling is easier and more straightforward. Your research project can progress more rapidly.

2. For what applications can the GEArray be used?

Use the GEArray to:

  1. Characterize gene expression profiles associated with experimental treatments, disease or development stages, phenotype and other biologically relevant phenomenon.
  2. Monitor activation of a specific signaling pathway.
  3. Provide an easy-to-use alternative to Northern blotting and RNase Protection Assays for relative mRNA quantification.
  4. Confirm gene expression results obtained from high-density genome-wide microarrays before continuing with more tedious gene-by-gene RT-PCR verification.

3. How do I use the GEArray to study gene expression?

GEArray is typically used to compare the expression of many genes between a control sample and one or more experimental samples using RNA prepared from cells previously treated under your experimental conditions. A simple single-tube enzymatic process converts the RNA into a biotin-labeled probe using a sample labeling kit specifically formulated for the arrays. The labeled sample then hybridizes with the nucleic acid crosslinked to the nylon membrane support. After washing excess non-specifically adherent label, a chemiluminescent detection method based on alkaline phosphatase (AP) conjugated to streptavidin illuminates the presence of the remaining label specifically hybridized to the microarray spots. All of the hybridization, washing, and detection steps are performed in the same disposable plastic hybridization tubes used to ship and store the arrays.

Each spot on the microarray contains nucleic acid corresponding to a single gene. An accompanying Gene Table identifies the genes represented by the microarray according to the location of its spot. The amount of light generated by the enzymatic turnover of the special AP substrate is directly proportional to the relative amount of each message in the original RNA sample. After correcting for background and normalizing the data to a factor that corrects for systematic error (such as a housekeeping gene or the median value), ratios of the intensity values between microarrays represent the fold-change in gene expression between the corresponding original experimental conditions.

4. How can I use the GEArray to study signal transduction? What is the Signal Transduction PathwayFinder™ GEArray? What is the next step after using the PathwayFinder™?

A single experiment using the Pathway-Centric™ GEArrays can answer many key questions about signal transduction in your experimental system or during your favorite biological response. Each microarray allows any of these questions to be answered about a single signal transduction pathway. For example:

  1. Are all key members of a signal transduction pathway expressed by the model system?
  2. Is the signal transduction pathway activated or deactivated during the experiment?
  3. Does the experiment affect the expression of any members of the signal transduction pathway?

However, if you do not know too much about your system except that it involves signal transduction, another special type of GEArray ascertains which signal transduction pathway promotes the biological response under study. The Signal Transduction PathwayFinder™ GEArray monitors the activation of 18 different signal transduction pathways. The microarray represents several genes encoding downstream effectors whose expression responds at the transcriptional level to the activation or deactivation of those signal transduction pathways. Determining which genes change their relative signal intensity on the microarray reveals the pathway or pathways relevant to the experiment.

If any changes are found in the expression levels of these pathway marker genes, continue to study their corresponding pathways in further detail with the appropriate pathway-specific arrays. Perform a more systematic study of the pathway in your experiment by examining the expression not only of the marker genes but also of the rest of the genes in the pathway such as ligands, receptors, kinases, scaffolds, and transcription factors. For example, if you find changes in the NFkB marker genes (such as NOS2, NFkB and MYC) using the Signal Transduction PathwayFinder™ GEArray, you can use our NFkB Signaling Pathway microarray to study additional NFkB pathway-related genes.

5. How sensitive, reproducible, and reliable are GEArray results?
A number of different parameters define microarray sensitivity including limit of detection and linear dynamic range. The GEArray detects messages as rare as 3 copies per cell from one million cells including low abundant transcripts such as un-induced TP53, CDKN1A and BAX. They detect the presence of 10 fM (femtomolar) concentrations of labeled sample in the hybridization solution. These numbers translate into a mass ratio of 1:300,000 meaning that these arrays can detect the equivalent of one specific message among 300,000 irrelevant messages. We used these results to develop our recommendations for the minimum amount of input RNA required for performing an experiment: Only 1 microgram (or under some conditions even as little as 100 ng) of total RNA (or ten fold less mRNA) is needed to perform a microarray experiment. When preparing RNA from cell culture, one 80% confluent T-25 flask or 100mm dish usually contains this amount of RNA. If preparing RNA from tissue, 100 to 250 mg of tissue on average should yield enough RNA, depending on the type of tissue.

The GEArrays also demonstrate a two to three order of magnitude linear dynamic range allowing the detection of a large range of expression levels and two-fold or even smaller changes in relative gene expression.

GEArray results are very reproducible. The raw intensities of duplicate arrays agree with a correlation factor (R-squared) of 0.9935, and the intensities of replicate arrays agree with an average coefficient of variation (= standard deviation / mean) of 5-10 percent.

The GEArray results are also reliable and robust. Slight changes in the initial microarray conditions, particularly sample loading, do not affect the observed relative gene expression profiles. The profiles also exhibit good cross-platform validation and agree well with conventional RT-PCR results.

6. Why are alpha-32P-dCTP and biotin-16-dUTP recommended as opposed to other labeled nucleotides?

Radiolabeled dCTP is the most commonly used nucleotide for radioactively labeling probes. We have formulated the buffers in our Probe Synthesis Kits (Buffer B or BL) for the cDNA GEArrays to accommodate the use of the more popular nucleotide, dCTP. If you prefer to use another radiolabeled nucleotide (such as dATP), we can provide you with our RT Buffer. It is devoid of nucleotide, and we will give you instructions to reconstitute all four nucleotides to their appropriate concentrations. Biotin-16-dUTP for the cDNA GEArrays (or biotin-16-UTP for the Oligo GEArrays) is the only commercially available biotinylated nucleotide that works well in our array assays. We have tried other biotinylated nucleotides (such as biotin-14-dCTP) and have only obtained substandard results.

7. How should I prepare my RNA sample and how should I check its quality?

We recommend using one of two different RNA isolation methods. For messenger RNA, we offer our ArrayGrade mRNA Purification Kit (GA-002); however, we do not recommend this method for all cell lines or tissues. Using this kit, we have successfully obtained quality RNA from various stable human cell lines, and several mouse tissues also gave good GEArray results. We do not recommend using this kit to isolate mRNA from whole blood or any blood-derived cell line, primary or transformed. To isolate total RNA instead, we highly recommend using the RNeasy Mini Kit from Qiagen. This kit purifies RNA of sufficient quality for microarray studies with the GEArray from all biological sources.

To insure a successful GEArray result, the isolated RNA must meet specific quality control criteria. The RNA should be at concentration of at least 0.5 mg/ml for total RNA or at least 0.1 mg/ml for messenger RNA. The A260:A280 ratio of the RNA should be at least 1.8. Characterization of total RNA samples by agarose gel electrophoresis should yield sharp (not smeared) 28S and 18S ribosomal RNA bands having a band intensity ratio of 2:1. Messenger RNA should yield a smear centering around 2-4 kbp on a denaturing agarose gel. Any of the following observations indicate degradation of the RNA sample: a smearing of either of the 28S and 18S bands for total RNA, a decrease in the rRNA intensity ratio in total RNA, a lower molecular weight range for mRNA, or a ratio of A260/A280 less than 1.8. In these cases, perform your experiment and the RNA preparation again before proceeding with the array analysis.

Using RNA isolation methods other than the above recommendations, particularly Trizol or cesium chloride gradients, often leaves the RNA sample contaminated with enzyme inhibitors that reduce sample labeling efficiency. Such samples may be further purified using the spin columns in the Qiagen RNeasy Kits. We also recommend that first time GEArray users test the success of their labeling reactions using the protocols in the Troubleshooting Guide of the appropriate User Manual before continuing with hybridization.

8. What are some good stopping points in the GEArray protocol?

After probe synthesis, you may store the probe at -20 °C overnight. If using cDNA GEArrays, remember to denature the probe again before adding it to the hybridization solution the next day by thawing, incubating at 94 °C (or boiling) for 2-5 minutes, and then immediately chilling on ice.

After pre-hybridization, you may store the damp membrane at -20 °C overnight in its original plastic tube after washing the membrane twice with 2X SSC. Repeat pre-hybridization as described in the User Manual the next day or when ready to continue.

After chemiluminescent detection particularly if you need a different exposure or missed your chance to capture an image, you may store the damp membrane in its original plastic tube at 4 °C overnight. The next day, briefly rinse the membrane twice 3 ml Buffer G (AP Assay Buffer) and repeat the incubation with CDP-Star and exposures as described in the User Manual.

9. Can I use the cDNA GEArrays for species other than human or mouse?

Based on our experience and that of our current customers, the mouse cDNA GEArrays can be used to analyze rat RNA given the high degree of identity between genes from the two different species. Some of our current customers have successfully used our human cDNA GEArrays to analyze RNA from a number of different species, such as pig, hamster, rabbit (Ning, XH, et al. (2002) J. Appl, Physiol. 92: 2200-2207.), monkey or other primate species (Bostik P, et al. (2004) J Virol. 78(3): 1464-72.). However, the probe synthesis and hybridization conditions must be optimized for your individual experiment.

We recommend using our RT-Labeling Kit (L-01) instead of either of our other sample labeling kits. During that protocol, we also recommend using random primers (Promega Catalog Number C1181) and/or oligo-dT primers (Promega Catalog Number C1101) instead of our gene-specific primer mix, Buffer A. Start with final concentrations of 25 µg/ml for the random hexamers and/or 50 µg/ml for the oligo-dT primers. The hybridization temperature and/or the stringency of the washes should also be decreased. Try lowering the hybridization and washing temperatures from 60 to 55 °C, and try washing with each washing solution once instead of twice. Continue to adjust these conditions until you achieve satisfactory results.

The accuracy of the experimental results varies from gene to gene and depends on the degree of identity between the individual genes of the two species. Therefore, relative gene expressions should always be verified by an independent and species-specific method (such as Northern or RT-PCR analyses using species-specific probes or primers) after performing these cross-species hybridization experiments.

The Oligo GEArrays are very species specific, and we do not recommend these microarrays for cross-species hybridization experiments. However, Oligo GEArrays specific for human, mouse, and rat are available.

10. Can I strip and re-probe the GEArray membrane?

Even under the best conditions, we observe a 60 percent loss of signal after stripping and re-probing our GEArrays despite the fact that we UV-crosslink the DNA elements onto the nylon membrane. We also find that individuals vary widely in their ability to obtain successful results from stripped and re-probed GEArray membranes. Therefore, we do not recommend stripping and re-probing the GEArray membranes to obtain publishable results and only recommend using the GEArrays once. However, the membranes can be stripped for troubleshooting purposes or to obtain preliminary results to be later verified with a new experiment using a fresh array membrane. Strip the membranes using a procedure similar to stripping blots used for Northern or Southern hybridization: Boil for 5 to 10 minutes in 0.5 % SDS, cool for 10 minutes, and rinse twice with 2X SSC. Store the damp membrane at -20 °C in its original plastic tube overnight if needed.

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