Microarray Research Section
Frequently Asked Questions
Who can order the Microarray Research Facility (MRF) mircroarrays?
MRF microarrays are directly available to investigators in the NIAID Division of Intramural Research or the Vaccine Research Center.
What is the sensitivity of the microarrays?
Sensitivity is dependent on the labeling and detection methods used. Obviously, those who use amplification techniques to label the samples will achieve much better sensitivity over the standard labeling methods. Conversely, the amount of RNA you can isolate dictates the type of labeling method or amplification method you will be able to use. In general, the amount of effort and experimental risk increases as the amount of RNA starting material decreases. There are published reports describing the use of extremely limited RNA for doing microarray analyses, but these methods may require some dedicated development time within your lab to get them working properly. See types of labeling methods for more information. Another point to remember is that sensitivity can vary with every labeling reaction and hybridization, so performing replicates is important.
How many cells do I need to conduct a microarray experiment?
If we assume that one can isolate 1 µg of total RNA per 100,000 eukaryotic cells, then one can calculate how many cells are needed for each of the sample labeling strategies. Briefly, direct cDNA and amino allyl indirect labeling require 2 million cells; amplified RNA requires 100,000 cells, and Nugen’s isothermal amplification method requires 1000 cells.
What types of sample labeling methods should I use?
We have in-house protocols for each of the labeling methods listed in the Protocols section. For investigators who are starting microarray programs, we recommend choosing one of the commercial kits that are available for these applications. Several vendors offer kits that are appropriate for these approaches and for situations where more amplification is required.
| Method |
Total RNA Recommended |
Summary |
| Direct cDNA |
10 to 30 µg |
Fluorescent nucleotides incorporated directly into cDNA during reverse transcription. |
| Amino allyl cDNA |
20 µg |
Amino allyl nucleotides incorporated into cDNA during reverse transcription; fluorophore coupling and clean-up are done subsequently. |
| Amplified RNA |
1 to 2 µg |
Double-stranded cDNA is the template for in vitro transcription that incorporates amino allyl nucleotides into aRNA; fluorophore coupling and clean-up are done subsequently. |
| Amplified cDNA |
5 to 100 ng |
NuGEN technologies offers a novel amplification kit for this application. Amino allyl nucleotides are incorporated into cDNA during amplification; fluorophore coupling and clean-up are done subsequently. |
For more information, view the Labeling Comparison Charts. (56KB PPT)
How do I get my results?
The MRF has two Axon GenePix 4000B scanners in Building 50 and one in Twinbrook I that are available to certified NIAID investigators. The GenePix Pro software is used to extract the data from the scanned images of the microarrays. The GenePix Pro software runs on the Windows operating system. For those who want to extract the data in their own laboratories, a license can be obtained through the purchase of a pass-through security dongle that plugs into the parallel port. Interested investigators should contact Mike Wilson, Ph.D.
For investigators who take advantage of the hybridization service, data can be retrieved from our WWW server after it is scanned. Investigators are responsible for managing their own raw data from the scanners. Most investigators will want to load their results data into the mAdb database.
What are the different file types associated with the genepix scanners?
There are five different types of files in GenePix. You will have six files saved for each of your arrays.
GAL GenePix Array List File (*.gal)
A GAL file is created for each print layout of an array and it is accessed through the mAdb Web site. The GAL file contains a layout of the array, the organization of blocks and features with a list of all the genes and their position. Instructions for saving GAL files to your computer are given in the mAdb. It will be necessary to download a GAL file for each version of arrays that are produced. The GAL file works in concert with the GPS file.
TIFF Tagged Image File Format (*.tif)
(Raw data images for analysis) After scanning an array, the image acquired in GenePix is saved as a TIFF file by default. This is an uncompressed graphics file format and it is quite large. This file is required for analysis because it contains the primary data of the single wavelength images saved from the scan in a multi-image TIFF file. DO NOT LOAD THIS FILE INTO THE mAdb.
GPS GenePix Settings File (*.gps)
The GPS file stores the positions and sizes of the grid of blocks and features (spots) that correspond to the array, as well as the settings for acquisition, analysis, and display. During analysis, the grid of the blocks and features is aligned with the array prior to extracting data. Save the alignments you make as you go along in a specific file (GPS) for each array. You will end up with the complete feature characterization for each scanned array in its own GPS file.
GPR GenePix Results File (*.gpr)
After griding the array and characterizing the individual features, GenePix Pro extracts the numerical results table that is saved as a GPR file. In GenePix Pro 4.0, this file is saved under the save results as line of the file saving menu. THIS FILE IS LOADED INTO THE mAdb.
JPEG Joint Photographic Experts Group (*.jpg)
Images for presentations that cannot be used for analysis. The default check box tells GenePix Pro to save the JPG files when the results file (GPR) is saved. GenePix Pro 4.0 saves three separate JPG files: one for each channel and the composite color image of the ratios. The two single-channel JPEGs have the ending *_W1.jpg, and *_W2.jpg, the composite image ends with _R1.jpg. LOAD ONLY THE COLOR COMPOSITE IMAGE FILE (*_R1.jpg) INTO THE mAdb.
What file types get loaded into the mAdb system?
Load the GenePix results file (*.gpr) and the color composite jpeg image (*_R1.jgp).
Where can I find a list of the genes on the array?
Gene lists for each microarray are available through the mAdb system.* If you do not find the gene you are interested in, try using NCBI's LocusLink to find the UniGene ID and perform your search on that identifier.
How do I know when I have the correct PMT settings for scanning?
Investigators can adjust the photomultiplier tube (PMT) prior to scanning to balance the two channels. There are some important principles to keep in mind with respect to this concept:
- A well-balanced labeling and hybridization is far more important than corrections done at the time of scanning. A doomed hybridization is never rescued with PMT adjustments.
- Normalization techniques during analysis will compensate for most minor channel imbalances.
- The goal of changing the PMT settings is to maximize the dynamic range of each channel while minimizing the number of spots with saturated pixels.
How do I place the coverslip in the correct position for hybridization?
Placement of the coverslip is only an issue for those who choose to do their own hybridizations, as the hybridization service takes care of this phase of the experiment. Placement of the coverslip over the exact array area on the slide is guided by a microarray template. One can place this sheet directly under the slide or the translucent hybridization chamber to direct placement of the coverslip.
What is the meaning of the codes printed on the label of each microarray?
Our standard microarrays are given an identification sticker that describes each microarray and helps the investigator orient the slide. Each slide is given a barcode that helps us automate data storage and tracking of arrays. The 8-digit unique array identification encodes the species, array version, lot number, and slide number. This provides a convenient, unambiguous nomenclature to help investigators track their hybridizations and results.
Several journals are now requiring microarray data to be submitted to a data repository. What is this and what is required?
The Nature journals now require that all microarray data be deposited into a repository to allow others to retrieve or analyze the data. The concept is similar to that required for sequence data submissions into GenBank. There is a convention emerging for the types of information required that is called MIAME: the Minimal Information About a Microarray Experiment. MIAME is proposed by the Microarrray Gene Expression Data Society (MGED).*
What is required to be MIAME compliant?
Some databases that allow direct submission of microarray data are said to be MIAME compliant. A checklist of the features of MIAME-compliant data can be found at MGED.* The MRF staff and developers of the mAdb are working together to add MIAME-compliant functionality to the mAdb. In the meantime, mAdb staff can assist investigators in importing their data to GEO (Gene Expression Omnibus),* which is the NCBI repository for microarray data. Submitting microarray data to GEO satisfies the requirements of most journals.
*Note: Some of the links on this page connect to information sources outside of NIAID and are provided as a convenience for World Wide Web users. Please see the NIAID disclaimer.
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