This video was produced by researchers at the University of Georgia's Warnell School of Forestry and Natural Resources. The purpose of this training video is to detail how to process our LOB lolly pine pt, gen two CDNA, microarray paying particular attention to the washing and handling of the array, both before and after hybridization. In general, our protocols follow those developed at the Institute for Genomic Research Tiger and the Vanderbilt Microarray Shared Resource Facility, VMSR, where our arrays are printed.
We have found that more stringent processing of PT Gen two is required to provide the greatest amount of consistency with respect to uniformity of signal quality and low backgrounds. The first step is a pre-wash. We do this because our spotting buffer contains a very high salt content and this pre-wash is necessary to remove that salt.
The slides are placed in a microscope slide carrier and plunge vigorously 15 to 20 times in a 0.2%SDS solution that has been preheated to 43 degrees. After the slides are washed in the SDS solution, they're removed carefully with forceps into a Copeland jar containing pre hybridization buffer. This buffer contains five XSSC 0.1%SDS, and 1%BSA, and is also heated at 43 degrees.
The Copeland jar is covered with parfum and placed in a 43 degree incubator for one hour. Following pre hybridization, the slides are removed to a microscope slide carrier and processed successively through five changes of deionized water plunging 15 to 20 times in each change. Finally, the slides are placed in isopropanol and dipped five or six times before being centrifuged to dryness in a chip M mate centrifuge.
After centrifugation, the slides are blown with compressed air passed through a 0.2 micron filter. The slides are then overlaid with lifter slips, which have also been cleaned with compressed air, and we use a small pipette tip to adjust the lifter slip into the correct position. On the slide.
After the lifter slips have been positioned, but before the hybridization buffer is added, we add 20 microliters of 100 millimolar dihi, three atol to the humidity wells located at the ends of the chamber. Now we're ready to add the target CDNAs, which have been resuspended in hybridization buffer. The pipette tip is placed at the edge of the lifter slip and the slide and the solution is slowly pipetted.
We found that utilizing the capillary effect to load the arrays provides the most consistency and greatly reduces the incorporation of any unwanted bubbles. After the target material has been added to the array, the hybridization chamber is assembled and covered with aluminum foil. The hybridization chamber is then placed in a shaking water bath for overnight incubation, typically 14 to 16 hours.
The water bath is set at 48 degrees and the shaker is set at 50 rpm. It's important to note also that from the point of addition of the hybridization solution through all post hybridization washes, the procedures are carried out in low light to prevent photo oxidation. The following day, the slides are removed from the hybridization chamber and placed in a Copeland jar containing wash solution number one, which has been preheated to 53 degrees.
The slides are allowed to remain in the Copeland jar for about a minute until the lifter slips fall away, at which point the slides are gently removed with forceps and placed in a microscope slide carrier to proceed with the washes. The slides are then plunged vigorously about 15 to 20 times in a second container, also containing wash solution number one at 53 degrees. The container is then covered with parfum and placed in an incubator air shaker set at 53 degrees and a hundred RPM.
After the 10 minute incubation and wash solution, number one, the slides are plunged vigorously 15 to 20 times into a container with wash solution number two, which is at room temp the container. This is been covered with para film and placed again on a shaker at room temp for a 10 minute incubation. Finally, the slides are removed from wash solution number two and plunged vigorously 15 to 20 times into a container with wash solution number three.
The slides are then removed into a second container of wash solution number three covered with perfil and again placed on the shaker for 10 minutes. After the final wash, we centrifuge the slides to complete dryness. To do this, we place a 15 mil Falcon cap into a 50 mil conical tube, and we place the arrays barcode side down into the conical tube.
The tubes are then spun in a swinging bucket head at 1500 RPM at room temperature. After centrifugation, the slides are removed to a second set of 50 mil conical tubes, which have been covered with aluminum foil. The tubes are then purged with nitrogen gas, which is filtered through a 0.22 micron filter and cap for transport or for storage until scanning.
We collect scan data on a PerkinElmer scan array 5, 000. The array image is gridded and raw scan data is processed and initial data analysis is done with bio discovery's imaging software suite. Next, the spot signal intensity data are normalized as statistical analysis are performed with BRB array tools, a robust and freely available microarray statistical package that can be downloaded from the National Cancer Institute.
Other statistical analysis and gene expression pattern searches are performed with the gene expression pattern and analysis suite Jeep PPAs. Another freely available web-based analysis package. Thank you for watching our video on how to process the lob lolly pine PT Gen two microarray.
The following individuals Were responsible for the content and production of this training video.
