Custom cDNA microarrays

The development of a custom cDNA library implies full collaboration with our customers. Once the contents of the library have been characterized by sequencing, we select and rearrange the clones that the customers have decided to include in the microarray. Our facility uses its high-throughput production and purification capacities to produce and control the PCR probes. Quality of the amplicons is rigorously controlled to ensure best microarray quality. Finally, once the locations of the cDNA probes on the slide glass are determined, printing can begin. The history of sample movement is tracked throughout the re-array, amplification, cleanup and final consolidation, using an automated barcode schema and custom-designed tracking software.

Some examples of custom cDNA microarray projects are Atlantic salmon, Spruce, Poplar, Rainbow Trout. These microarrays are the property of the institutions that funded the projects.


Probe production

Our facility has the capacity to produce custom cDNA probes whereby the cDNA of interest is amplified and printed. The probe production process involves rearray, amplification, purification, quality control (QC) of purified products, and consolidation into printing plates prior to printing. The following outlines the probe production process.

Rearray

We have the capability to perform needle innoculation "cherry-picking" rearray. Clone lists are supplied by the customer along with the source plates. The source plates can be either 96 or 384-well plates and the destination plates produced are 96-well plates. The rearray is performed on the BioRobotics TasII microarray robot in a HEPA-filtered, humidified and chilled environment. The destination plates are also replicated and stored for further assessment and/or as archival material.

Rearray Quality Control

Rearrayed clones are grown up in antibiotic selective media and the turbidity assessed by spectrophotometer. This represents a quality checkpoint for the clonal growth - any clones with poor growth can be regrown and/or rearrayed. Also, 1 clone from each 96-well daughter plate is sequenced as a verification of both the daughtering (replication) process and the rearray process.

PCR

Plasmid inserts are amplified from the re-arrayed parent plate using vector specific primers. Amplification is done in 96-well plates using the BioRad Tetrad Thermocyclers.

Nucleic Acid Purification

Amplicons are purified using the Qiagen Biorobot 3000 system, a versatile liquid handling workstation designed to automate the purification of nucleic acids. The Biorobot 3000 series is equipped with a high speed pipetting and tip change system which provides reliable pipetting and high-throughput purification, with minimal hands-on time.

Amplicon Quality control

The Labchip 90 system is used to assess the amplicon quality either prior to or post Qiagen clean up. This system is based on microfluidics technology, giving amplicon sizing and concentration in just 30 seconds per assay. We strive to be at a printing concentration of 200ng/ul based on the Labchip 90. The PCR products are randomly sequenced for verification purposes. During this phase, frameshift errors that arose during re-array will be discovered. The Tango and the Qiagen are used to consolidate the cDNA into 384 well printing plates. The products are lyophilized by the speed-vac and and resuspended in printing buffer.