Microarrays
Q. Where are the latest protocols for CodeLink™ microarrays?A. The latest protocols can be found at: http://www.appliedmicroarrays.com/CodeLink.html
Q. How does Applied Microarrays validate its microarrays?
A. Each whole genome array is inspected for spot presence and morphology, as well as any array defects. In addition, functional assays are performed on multiple slides from each manufacturing batch. For a batch to be released, the assay results must meet several predefined quality metrics in categories such as probes below noise, negatives above noise, correlation coefficient, and minimum fold change.
Q. How many features are on a human, mouse and rat CodeLink™ microarray?
A. The number of features and gene classes are located at the following URLs:
CodeLink™ Human Whole Genome Array: http://www.appliedmicroarrays.com/Human.html
CodeLink™ Mouse Whole Genome Array: http://www.appliedmicroarrays.com/Mouse.html
CodeLink™ Rat Whole Genome Array: http://www.appliedmicroarrays.com/Rat.html
Q. What type of quality control information do I get with each microarray?
A. A CD containing a Manufacturer’s Spot Report file is shipped with each microarray order. The MSR file contains quality control information specific to each array that you receive, determined from the individual slide inspection performed at post-dispense quality control. CodeLink™ Expression Analysis software is necessary to interpret the MSR file, and the information will be automatically incorporated into the analyzed data tables as a probe flag, along with any other quality flags generated during analysis to avoid the use of accurate data.
Q. Is one probe per transcript sufficient data within an array to provide statistically relevant data?
A. Since we have a high degree of precision within our microarrays (i.e. 99.5% of ratios within 2-fold for a typical array-to-array comparison) the answer we obtain from 1 measurement is consistent. Repeated measurements within an array have marginal if not insignificant benefit due to our high precision. The highest degree of variability is seen across different production batches of arrays, user-to-user assay differences, typical day-to-day variability, scanner variability, etc. Therefore, scientists using our arrays will benefit the most by replicating at the technical, experimental, or biological level.
Q. If I consistently spike the positive controls at the cRNA level following the procedure described in the Application Notes, can I use the linear curve generated by the positive controls to normalize between different slides?
A. It is not advised to normalize against such a small subset of controls. In addition, cRNA 'spiking' normalization does not take into account the remainder of the cRNA, which could be of poor quality. The purpose of the cRNA 'spiking' is to indicate whether the bioarray and post in vitro transcription assay worked properly. For whole genome arrays, normalization through use of the overall slide median is highly recommended for the most reproducible array data, and this normalized data will be generated automatically if this normalization is selected in the CodeLink analysis software. For smaller focused arrays, other normalization options exist within v5.0 of the CodeLink software, including the use of an integrated housekeeping gene set on each microarray, as well as a user-defined set of probes from the array.
Q. Why do you offer 55K probes on the CodeLink™ Human Whole Genome array while we assume there are only 35K human genes?
A. Sequences are targeted to transcripts rather than genes. One gene can generate more than one mRNA transcript version, through the use of alternate start sites, end sites, and alternative splicing.
Q. What is the expiration date on the CodeLink™ activated slides?
A. The expiration is a minimum of 2 months from the date the arrays are received by the customer. Expiration dates are included on the bioarray pouch packaging for your reference. If you have specific expiration or lot # requirements, we will do our best to accommodate your needs. Please send your requests to This e-mail address is being protected from spambots. You need JavaScript enabled to view it .
Q. What are the dimensions for the CodeLink™ microarray slides?
A. The array dimensions are 14.25 x 53.64mm. These are the nominal dimensions. The design tolerance is +/- 0.05mm.
Q. What is the total time necessary to run CodeLink™ microarrays?
A. It takes about 2.5 days to run a complete bioarray assay with traditional mRNA amplification methods. The first day is dedicated to first-strand cDNA synthesis, second-strand synthesis, purification of double-stranded cDNA, and then an overnight in vitro transcription. Although this is a full day of experiments, there are two 2-hour incubation periods. During the next morning, the cRNA is purified and later that afternoon the fragmentation and hybridization reactions are set up. The second day is not a full day of experiments and consists of a couple of hours in the morning and a couple of hours in the afternoon. The third day is devoted to washing/staining for about 2 hours and then bioarray scanning and data analysis.
Q. What scanners are compatible with CodeLink™ Expression Analysis Software?
A. For accurate quantification of spot intensities of the Applied Microarrays whole genome and catalog focused arrays, the scanner used needs to have a 5µm pixel size capability. For a list of scanners that have been validated with CodeLink™ Expression Analysis Software, please see the document: Reagents & equipment for CodeLink™ Microarrays. For custom bioarray products, please inquire on specific scanner requirements.
Q. Why do you recommend using Cy5 dye instead of Cy3?
A. We recommend using Cy5 dye instead of Cy3 is because the Cy5 (red channel) is brighter with less background. Although Cy3 is more stable, it results in a higher level of background signal; this is because the green channel has the ability to pick up signals from dust and other contaminants, salts, slide substrates, and other autofluorescing materials.
Q. What is the difference between biotin-11-UTP vs. biotin-16-UTP?
A. Biotin-16-UTP has a longer linker arm for the biotin label. It has better detection sensitivity since the biotin is more accessible by streptavidin.
Q. Can I directly labeling my target with Cy5 or Cy3 dyes?
A. This is not recommended as a standard process. In our experience, Cy5 or Cy3 directly-labeled target results in a high bioarray background, thereby reducing the signal-to-noise level achieved. If this type of labeling is required for a particular application, the user will very likely be required to modify the recommended CodeLink bioarray hybridization and/or wash conditions to achieve a comparable slide background, and therefore the expected signal-to-noise level.
Q. What type of centrifuge is recommended for the CodeLink™ bioarray drying step ?
A. The required dimensions are a depth of 2 inches, length of 5 inches, and width of 3.5 inches. The Sorvall Superspeed T-21 centrifuge will work with the CodeLink™ reservoirs. You will need to use the ST-H750 rotor (according to Sorvall this is the standard rotor) with the 11779 microplate carrier.