प्रसंस्करण विनोदी पाइन PtGen2 सीडीएनए माइक्रोएरे
Summary
सीडीएनए माइक्रोएरे PtGen2 जीन अभिव्यक्ति के अध्ययन के लिए विनोदी पाइन में विकसित किया गया था,<em> पी. taeda</em> और अन्य शंकुवृक्ष प्रजातियों. यहाँ, हम बताते हैं पूर्व और पोस्ट – संकरण हैंडलिंग और तकनीकों है कि इस सरणी के साथ इस्तेमाल किया जा सकता है बेहतर स्थिरता, कम कलाकृतियों, और कम पृष्ठभूमि उपज धोने.
Abstract
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.
Protocol
Discussion
PtGen2 एक हाल ही में विकसित की है, कस्टम सीडीएनए माइक्रोएरे है कि मुख्य रूप से विनोदी पाइन अनुसंधान समुदाय द्वारा इस्तेमाल किया जा डिजाइन किया गया था है. प्रारंभिक उत्पन्न परिणाम इस प्रकार दूर सरणी से पता ?…
Acknowledgements
लेबलिंग, संकरण, और धोने प्रोटोकॉल के साथ साथ जबकि जीनोमिक अनुसंधान (अधिक पढ़ें), Vanderbilt माइक्रोएरे साझा संसाधन (VMSR) में डा. शॉन लेवी के लिए संस्थान में डॉ. जे Quackenbush द्वारा पहले विकसित उन लोगों के लिए कुछ संशोधनों के, और डा. होते रोब अल्बा जबकि Boyce थॉम्पसन संस्थान (BTI) कार्नेल विश्वविद्यालय में.
Materials
| 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 |
References
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- Lorenz, W. W., Dean, J. F. D. . unpublished data. , .
- Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Hughes, J. E., Snesrud, E., Lee, N., Quackenbush, J. A concise guide to cDNA microarray analysis. Biotechniques. 29, 548-562 (2000).
- Alba, R., Fei, Z., Payton, P., Liu, Y., Moore, S. L., Debbie, P., Cohn, J., D’Ascenzo, M., Gordon, J. S., Rose, J. K. C., Martin, G., Tanksley, S. D., Bouzayen, M., Molly, M., Jahn, M. M., Giovannoni, J. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development. The Plant Journal. 39, 697-714 (2004).
- Lorenz, W. W., Simões, M., Miguel, C., Dean, J. F. D. Analysis of Gene Expression changes in Pinus species using a Loblolly pine cDNA microarray. IUFRO-CTIA 2008 Joint Conference. , (2008).
- Simões, M., Lorenz, W. W., Alba, A., Gonçalves, S., Dean, J., Miguel, C. Global gene expression analysis during P. pinaster embryo development. 7th Plant Genomics European Meeting. , (2008).
