Protocols
Commercial kits often provide convenience and high-quality reagents for creating labeled cDNA or amplified RNA targets. Users are encouraged to peruse the table in the Resources section.
RNA Amplification Methods
The Microarray Research Facility (MRF) and several other investigators are obtaining successful results using RNA amplification techniques. We typically see 200- to 1000-fold amplification of the mRNA subset through a single round of the procedure. A protocol for amplifying and labeling RNA is available. Alternatively, several commercial kits are available for amplifying RNA and/or labeling the amplified RNA for hybridization to microarrays.
The important features of the amplification procedure are outlined below. Amplified RNA (aRNA) is produced by in vitro transcription from a phage promoter. This aRNA matches the complementary DNA strand—it is also commonly referred to as cRNA. The aRNA is labeled and hybridized directly to the probes on the array. Users should pay close attention to RNase-free techniques throughout the procedure.
Labeling of the aRNA (56KB PPT) is typically performed in a two-step process. First, an amino-allyl UTP is incorporated into the polymerized aRNA molecules during in vitro transcription. After cleanup, the incorporated primary amines are reacted with a monofunctional fluorescent dye (typically NHS esters of either Cy3 or Cy5) and any unreacted amines are blocked. The freshly labeled aRNA molecules are cleaned again and prepared for hybridization. This entire procedure is more time consuming than the conventional cDNA labeling, and it requires strict RNase-free handling techniques for all phases of the preparation.
Training
- Users must be trained and certified before using Axon's GenePix 4000B fluorescent scanner. Training on the scanner software is required to enable users to convert the raw pixel data into numerical output that is suitable for analysis.
- New users can also receive hands-on training on the techniques of microarray hybridization and probe labeling. Contact Timothy G. Myers, Ph.D., to arrange training.
- The Center of Information Technology (CIT) offers regular training courses on how to use the mAdb system* for data archiving and analysis. Advanced training on using the specialized BRB array tools is also arranged through CIT.
Data Analysis
- The Data Analysis and Bioinformatics group can assist investigators with questions about experimental design and strategies for microarray data analysis.
- MRF works closely with John Powell's group at the NIH Center for Information Technology (CIT). The CIT group is implementing the NIAID version of the mAdb database and analytical tools. NIAID investigators should link to the mAdb system* and register as a user of this system in order to gain access to the database. CIT offers regular training courses on how to use the mAdb system for data archiving and analysis.
- MRF uses Axon's GenePix Pro software for extracting the data from the scanned images of the microarrays. Data extraction from the scanned images can be performed in MRF; however, investigators who require scanning take priority over anyone who is performing image analysis. The GenePix Pro software runs on the Windows environment and the license is enforced through the purchase of a "security dongle" that plugs into the USB port. Interested investigators should contact Tim Myers for more details.
- Silicon Genetics' GeneSpring: Floating site licenses are available for GeneSpring. Contact Tim Myers for more details.
Experiment Design Tips
Designing a microarray experiment is very important for generating results that can be interpreted with confidence. These tips are just a few of the considerations that go into planning a microarray-based project, so anyone who is contemplating a major study should contact Tim Myers.
Replicates Are Essential
Estimating the significance of results with the associated confidence limits requires 4 to 5 replicates. Of course, the power of the analysis will improve with more replicates. We generally recommend that investigators perform 4 to 5 replicates initially in order to estimate the level of noise that will be part of the experimental system. After that, investigators are instructed to estimate the power of the analysis and determine if more replicates are needed.
Appropriate Reference RNA
A universal reference RNA is the same RNA stock that is paired with each test sample during hybridization. The metric of comparison will be the ratio of experimental-to-reference samples for each probe. A problem arises when the reference sample does not have a measurable signal, resulting in an erroneous ratio. For this reason, an appropriate reference sample is sometimes a pool of RNA from all the test samples that will be included in the project.
Track Potentially Confounding Variables
There are always opportunities to confound a microarray experiment because many of the processing steps can introduce systematic errors. Sources of confounding variables include different microarray lots, different sources of the experimental sample or purified RNA, different people doing the labeling and hybridization, or labeling and doing the hybridization on different days. The best approach is to control these effects using proper experimental design. Investigators should track values of potentially confounding variables so that their effects can be estimated or otherwise considered in the final analysis.
Latest News on the mAdb Web Site
For the latest information about array developments, visit the mAdb system.* Contact mAdb for new information about hybridization techniques, controls, and more.
*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.
back to top