Saturday, November 20, 2010

Microarray Hybridization - Manual versus Automation

Various hybridization tools and machines exist to perform the different tasks of microarray processing in the microscope slide size format. Tasks typically performed in this order are:

1. pre-hybridization
Prepares microarray for hybridization. Surface chemistry is blocked to prevent unspecific binding to the microarray, prevents background problems.
2. wash
Remove all reagents used in pre-hybridization to leave a pristine microarray ready for hybridization. In some cases microarrays are dried at this step and stored clean and dry until hybridization. Microarrays stored for long periods of time should not be processed.
3. hybridization
Perform specific binding reaction, maintain temperature throughout, prevent reaction from drying, achieve an even distribution of molecules in solution across the microarray for the duration of the reaction.
4. wash
Remove hybridization reaction and set stringency to wash to remove unbound biomolecules from the microarray.
5. dry
Get an even dry so the microarray can be scanned or imaged.

Manual processing of microarrays is most common. The hand operated tools to process microarrays manually are in-expensive and with only a little practice very good results are obtained. Hybridization Cassettes, Wash Stations and Drying tools are robust, simple and easy to use tools. Over the years have have sold thousands of these tools. They are standardized to the microscope size slide substrate and can be re-used many times, thus adding to their utility.

However, like all laboratory processes, microarray processing is becoming more automated. There are several commercial hybridization stations available. The most sophisticated of them provide all the thermal, hybridization, wash and dry processes to make a microarrays ready for scanning. Others perform only the hybridization using specialized cover slips that mix low volumes of liquid using diaphragms. Diaphragm pumping of liquids back and forth over the microarray is the most common method of mixing hybridization reactions. Most instruments allow at least 2 microarrays to be processed and others run up to 48 by connecting multiple processing units to a single computer. Individual ports allow for independent hybridization cocktail introduction for each microarray.

Why use a machine if manual tools work fine?
The reasons are:

  • To minimize handling of microarrays, thus reducing the possibility of human error.
  • Get better control over the experimental variables, resulting in increased reproducibility
  • Increase kinetics to reduce hybridization times
  • Empower users to define, edit and store individual methods, protocols
  • Save and link experimental procedures to databases
At ArrayIt we provide the only microarray hybridization station based on chaotic advection mixing, the system, pictured above is called TrayMix. This is an excellent mixing method and does a great job of increasing kinetics and reproducibility. More sophisticated than diaphragms, chaotic advection ensures that the target in solution equally addresses all probe and target interactions on the microarray. The temperature in the system is regulated from ambient to 75 °C +/- 0.1°C. Upstream from the reaction area which includes the mixing loop and hybridization chamber, a manifold introduces a programmable 5-way solution selection. Pre-hybridization, hybridization buffer, washing solutions and decontamination solution can be programmed and run. To get information on the TrayMix2 hyb station that uses chaotic advection, click here.

To see a movie of how well the mixing loop works on the TrayMix 2 works and a critical analysis of all the hybridization mixing techniques please read my other blog entry on microarray hybridization by clicking here.

Other reasons to stay manual...
Most hybridization stations do not allow multiple microarrays to be hybridized on a single slide. If they do, the current maximum is 4. All hybridization stations can be engineered to do more, contact me with your needs in this area. For low density microarrays, formats of 16, 24, 48 and 192 microarrays per slide formats are quite useful and high throughput. Clearly the more microarrays on a slide, the more difficult it becomes to automate. We are developing new slides that will allow for twelve 1ul hybridization reactions on a single slide using our standard hybridization for information on this new tool.

When choosing a hybridization method, consider the size and density of your microarray, your throughput requirements, and budget. If you are on a tight budget, manual is the choice. If reproducibility is most critical and you have the budget, go for automation. If you have many low density microarrays to process, consider the tools that make 24 microarrays per slide and puts them into a tool for 96 well processing on a liquid handling system, Arrayit Catalog ID: AHC4x24.  Manual or automated, we have many choices to fit the need. If you would like to discuss your application, do not hesitate to contact