The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.
PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing.
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips™ are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48°C in high humidity conditions. After hybridization, a series of standard washes are done at 53°C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.
(Note Sections 1 – 4 Not Demonstrated in Video)
Preparing Cy-Labeled Target cDNA
What follows is the protocol we use for cDNA synthesis from loblolly pine total RNA and indirect cDNA labeling prior to microarray hybridizations. Although a few non-trivial modifications have been made, the protocol below is similar to that in the instruction manual provided with the Invitrogen’s SuperScript Indirect cDNA Labeling Kit.
Part 1: First-Strand cDNA Synthesis Reaction
Part 2: Purifying First-Strand cDNA.
Part 3: Labeling with Fluorescent Dye
When preparing the reaction, be careful to minimize exposure of the dye solution to light. Also, DMSO is hygroscopic and will absorb moisture from the air, which will react with the NHS ester of the dye and significantly reduce the coupling reaction efficiency. Keep the DMSO supplied in the kit in an amber screw-capped vial at –20°C, and let the vial warm to room temperature before opening to prevent condensation. Use only the DMSO provided with this kit.
Part 4: Assessing the Labeling Procedure.
Part 5: Slide Pre-Wash
Part 6: Pre-Hybridization
Part 7: Hybridization
Should be carried out in the dark or low light.
Part 8: Post-Hybridization Washes
All washes should be carried out in the dark or low light. Slides should never be allowed to dry until the final step.
Part 9: Data Collection and Statistical Analysis
Figure 1. Example of PtGen2 array processed using this protocol.
Figure 2. Example of PtGen2 array processed using standard hybridization and washing protocols.
PtGen2 is a recently developed, custom cDNA microarray that was designed to be used primarily by the loblolly pine research community. Preliminary results generated thus far have shown the array to be a valuable tool in the evaluation of transcriptional events that occur in response to drought stress, as well as in monitoring changes that occur during embryo development in maritime pine, Pinus pinaster 5,6 We have also recently demonstrated that PtGen2 works well in cross species hybridizations using target samples isolated from a diverse range of conifer genera.5 As PtGen2 becomes more utilized by researchers studying gene expression in Pinus and other conifer species, this training video should provide them with the necessary technical foundation to generate quality and reproducible data. In our hands, PtGen2 yielded better results when processed by more stringent hybridization and washing protocols than those typically employed in microarray work. Other approaches to processing the PtGen2 array have been successful but lacked consistency. Following the technical steps described here will help to increase consistency, particularly when processing large numbers of arrays, and will also help to greatly reduce and eliminate unwanted artifacts and high backgrounds.
The labeling, hybridization, and washing protocols herein contain some modifications to those developed previously by Dr. J. Quackenbush while at The Institute for Genomic Research (TIGR), Dr. Shawn Levy at the Vanderbilt Microarray Shared Resource (VMSR), and Dr. Rob Alba while at the Boyce Thompson Institute, (BTI) Cornell University.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Pre-hybridization Buffer | Buffer | 5X SSC, 0.1% SDS, 1% BSA | ||
Hybridization Buffer | Buffer | 30% formamide, 5X SSC, 5X Denhardt’s, 1% PolyA RNA (Invitrogen, POLYA.GF), 0.1% SDS | ||
Wash Solution #1 | Buffer | 1X SSC, 0.2% SDS, preheat to 43°C | ||
Wash Solution #2 | Buffer | 0.1X SSC, 0.2% SDS | ||
Wash Solution #3 | Buffer | 0.1X SSC | ||
Isopropyl Alcohol | Reagent | Sigma Aldrich | 34959-2.5L | |
50mL Conical Tubes | Other | Falcon™ | 352070 | |
15mL Conical Tubes | Other | Falcon™ | 352196 | Use this or a similar cap placed at the bottom of each 50mL conical tube during centrifugation |
Coplin Jar | Wheaton™ | 900520 | ||
Staining Dish & Rack | Other | Wheaton™ | 900200 | |
Albumin from bovine serum (BSA) | Other | Sigma™ | A-9418 | |
PolyA RNA | Invitrogen™ | POLYA.GF | ||
SuperScriptTM Indirect cDNA Labeling | Kit | Invitrogen™ | L1014-02 | |
Turbo DNase | Kit | Ambion | AM1907 | |
System | ||||
Cy-5 Dye | Reagent | GE™ | PA25001 | |
Cy-3 Dye | Reagent | GE™ | PA23001 | |
LifterSlips™ | Other | Erie Scientific Company™ | 25X601 | |
HybChambers | Equipment | GeneMachines | JHYB200003 |