Processing the loblolly pine PtGen2 cDNA microarray.
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. 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. 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 degrees C in high humidity conditions. After hybridization, a series of standard washes are done at 53 degrees 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.
Lorenz
W Walter
WW
Warnell School of Forestry and Natural Resources, University of Georgia, USA. wlorenz@uga.edu
Yu
Yuan-Sheng
YS
Simões
Marta
M
Dean
Jeffrey F D
JF
D002232:Carbocyanines / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D018744:DNA, Plant / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D041603:Pinus taeda / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism
D002232:Carbocyanines; D018076:DNA, Complementary; D018744:DNA, Plant; D005456:Fluorescent Dyes; D018749:RNA, Plant; C089187:cyanine dye 3; C085321:cyanine dye 5