Reference Materials
Frequently Asked Questions
Software
Q. How do I know if I have the most recent software version? How do I get updates?
A. The CodeLink Expression Analysis Software icon on your desk should display the version of the software. You can also open the software, go to Help on the tool bar at the top of the screen and then go to About. The current version available from Applied Microarrays is v5.0. To obtain an updated version of the software, contact sales@appliedmicroarrays.com or your local distributor if you are outside of the U.S.
Q. What are the differences between the new software version 5.0 and the previous version 4.2?
A. The expression reports are more flexible; they can be cut and pasted into Microsoft PowerPoint, for example. More scanner options are available in the drop-down menu for version 5.0 and the updated software also provides more flexible gridding options. The ability to normalize using alternate methods, such as through the use of housekeeping genes and user-selected probe sets was also introduced. This is important for smaller, focused arrays.
Q. How do I find my Machine Code for registration of my CodeLink Expression Analysis Software?
A. If your CodeLink software version is not yet registered, you will see a message when you attempt to open the software indicating that the software is not licensed for use. A radio button will be available labeled Register. If you click on this button, a new window will open which will list your machine specific code.
At a later date after registration if you require the Machine Code, this can be accessed through the following process: Open the CodeLink Expression Analysis Software, go to Help on the tool bar at the top of the screen and then go to About. A window will appear, then click on Show Machine Code.
Q. What are the system requirements to run CodeLink software?
· Intel™ Pentium™4 or equivalent processor at 1.8 GHz
· Microsoft™ Windows™ XP Professional with Service Pack 1 or Windows 2000 Professional with Service Pack 2
· 1GB RAM (2 GB recommended)
· 1024 x 768 pixels in video display resolution
· 1.5 GB of available hard-disk space on the C drive
· Internet Explorer 6.0 or later
· CD-ROM drive
· Network interface card (NIC) or equivalent device for connecting the computer to the internet via a local area network (LAN)
Q. CodeLink software generated an error message saying that the program could not process the multipage *.tif format.
A. With regards to the batch data analysis, multi *.tif files cannot be imported into the CodeLink Analysis software. Please go to the scanner settings and ensure that it is not generating multi *.tif files (such as a dual color scan or a combination of preview and high resolution files).
Q. Can I perform dual color scans with CodeLink Analysis Software?
A. Yes, but each analysis needs to be saved as a separate file.
Q. Can we delete batches without deleting the image files used to generate the data?
A. Yes, you can delete them because you are just deleting the analysis information. You should still have the original *.tif image with your raw data where you stored them originally.
Q. When I try to analyze a Sample Image using CodeLink™ Expression Analysis Software, I get an error message indicating that it cannot decrypt the MSR file.
A. 1. Open the "Control Panel" folder
2. Run the "Regional and Language Options"
3. Is it using "English (United States)” as the standards and formats? Please use the English (United States) setting.
If this does not solve the problem, please send the following information along with a screen shot of your error message to support@appliedmicroarrays.com :
* Operating system and service package number. For instance, XP Professional SP3 (right click on My Computer, then select Properties).
* Regional and Language option.
Q. When I am creating a batch to export, I see an error message that says, “Please make sure all images have product information.”
A. Go to the following location on your computer’s hard drive:
C:\Program Files\Amersham Biosciences\CodeLink EXP v5.0\ProductFiles
This folder should be populated with files. If it is not, then insert the CD data disk that was shipped with your microarrays. View the contents of the CD and drag the Product Files from the CD-ROM into the file location listed above.
Q. I am using analysis software other than CodeLink Expression Analysis software and I need GAL files to assist in my analysis. How do I obtain the GAL files?
A. Contact support@appliedmicroarrays.com and we will send you the appropriate GAL files.
Reagents
Q. How do I know if the quality of my cRNA is appropriate for the CodeLink™ platform?
A. Quality assessment of the cRNA before fragmentation and hybridization is highly recommended. There are several methods for assessing cRNA concentration, purity and quality and they are described in greater detail in “Protocols for CodeLink™ Control RNA Spikes – Including tips for assessing RNA concentration, purity & quality”. The cRNA should have an A260:A280 ratio of 1.8-2.1. Using denaturing agarose gel analysis, amplified cRNA should appear as a smear from 250 to 5,000 nt. The average size of biotin-labeled cRNA should be approximately 1,400 nt. Using a bioanalyzer, the cRNA profile should display a distribution of sizes from 250-5,500 nt., with most of the cRNA between 1,000-1,500 nt.
Q. What are the storage conditions for CodeLink™ reagents?
A. 5X Fragmentation Buffer: 4°C
Hybridization Buffer Component A: Room temperature
Hybridization Buffer Component B: 4°C
Cy5-Streptavidin: Typically shipped at room temperature. Upon receipt, store at 4-6°C up to the point when you open the bottle. Hydrate contents of the bottle and make aliquots, then store frozen at -20°C.
CodeLink™ Microarrays: Room temperature in a dry environment.
Q. How much RNA starting material is required to perform a microarray?
A. Applied Microarrays recommends starting with 1 µg of total RNA in the in vitro transcription step of target preparation; however, this will vary by target preparation methodology used and also with the source and quality of the total RNA material.
10ug of prepared cRNA is used per CodeLink™ microarray for hybridization.
Q. Which reagents and equipment are necessary to run CodeLink™ microarrays?
A. Please see the document, Reagents & equipment for CodeLink™ Microarrays.
Q. Do I need to worry about RNase contamination when preparing my RNA samples?
A. Yes. Exercise caution to avoid RNase contamination. All solutions must be RNase-free. Pipette tips must be changed before each step. Use nuclease-free water for all nucleic acid steps. Use aerosol-resistant tips for all pipetting steps.
Custom Microarrays
Q. How do I place a custom microarray order?
A. Go to our Custom Array Quote Form at: http://www.appliedmicroarrays.com/ArrayDesignsQuotes.html . Supply the information that you know. We can advise on the best options for any details which you are unsure. We will rapidly produce a quote based on the provided details.
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 sales@appliedmicroarrays.com.
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.
Data Analysis
Q. How do I normalize my data?
A. Applied Microarrays strongly recommends whole array median normalization for all CodeLink whole genome bioarrays. Descriptions of the relevant calculation methods are below, along with a description of other software calculations exported in the data sets.
Median Normalized Intensity: Intensity is defined as the sum of the pixel signals in the area of interest minus the scaled 2-pixel median background. The intensities are then median-normalized by calculating the median of all spots (except for the control probes and blanks). Each spot intensity is then divided by the median value.
Negative Control Threshold: After median normalization, the negative control threshold is calculated using a set of predetermined negative controls. Negative control threshold = (20% trim-mean of negative controls) + 3*standard deviation using the trim mean set.
Coefficient of Variation (CV): CV = (STDEV/MEAN). CV is generally displayed as a percentage. For example, a (STDEV/MEAN) value of 0.08 corresponds to 8%.
Limit of Detection of Differential Expression: This is the calculation of the minimal detectable spot ratio that can be distinguished from platform noise. Only spots with intensities above the negative control threshold of the slide are used for minimum fold comparisons. The percent within 2 fold lists the percentage of spots having ratios less than 2 for each slide-to-slide comparison. The 95% within this fold result is the number at which 95% of the spot ratios fall below. Thus, the lower the variability and noise of the system, the closer the ratios will get to the value of 1.
The Log2 Ratio calculation: Differential expression is reported as the log2 ratio of two spot values. A spot with equal signals on each array would have a log2 ratio of zero, while a case of differential expression would result in a ratio value of 1 or greater. A log ratio of 1 represents a 2-fold change.
Q. Why are there negative numbers for the intensity?
A. The "raw intensity" is calculated by taking the Mean Intensity of the pixels within the Spot minus the Median of the local background which is computed from the periphery of the spot. If your spot has very little or no signal, the median of the local background may be slightly higher than the signal. This condition will generate a negative number. With regards to your analysis, if you encounter a negative number then you have little or no expression of that gene.
Q. How do I prepare my CodeLink™ data for GEO submissions?
A. Contact us at: support@appliedmicroarrays.com and we will provide you with a CodeLink™ Expression Analysis Software patch to download. Once installed, the patch will allow you to export in a tab-delimited text file for direct GEO submission.
