Processing the Loblolly Pine PtGen2 cDNA Microarray

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 (Note Sections 1 -4 Not Demonstrated in Video) Preparing Cy-Labeled Target cDNAWhat 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.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster

“…The hybridization reference sample consisted of a pool containing equal amounts of total RNA from all five time points investigated. Fluorescent labelling, hybridization, pre- and post-hybridization washes, scanning, and data processing were performed according to Lorenz et al [ 86 ], except that 2 μg of Cy-labeledamplified RNA was used to hybridize with the array after fragmentation using Fragmentation Reagent (Ambion, Cat. #AM8740).…”

Microarray analysis and scale-free gene networks identify candidate regulators in drought-stressed roots of loblolly pine (P. taeda L.)

“…Microarray processing and hybridization protocols have been described previously [ 36 , 45 ]. Slides were incubated in a HybChamber™ (Genomic Solutions, Ann Arbor, MI) containing 20 uL 100 mM DTT in the humidity wells.…”

“…Towards this objective we developed a collection of public genomics tools, including a set of ca. 172,000 Sanger ESTs from mostly root tissues, an annotated 26496-feature cDNA microarray for pine (PtGen2), and optimized protocols for microarray analysis of pine tissues [ 32 , 34 - 36 ]. After validating the PtGen2 microarray, we identified 2445 genes that are differentially expressed in drought-stress roots from P. taeda and demonstrated that the transcript population in P. taeda roots returns to the pre-drought state within 48 hours of cessation of the drought treatment.…”

Metatranscriptomic analysis of ectomycorrhizal roots reveals genes associated with Piloderma–Pinus symbiosis: improved methodologies for assessing gene expression in situ

“…The CTAB/LiCl method described by Chang and colleagues (1993) was able to recover good quality RNA from root samples of P. taeda; however, the quantity of recovered RNA was relatively low (∼ 6 ηg RNA mg −1 root cluster). Several CTAB methods modified from Chang and colleagues (1993) or Liao and colleagues (2004) were applied to extract RNA from larger amount of pine tissues (1-4 g) (Joosen et al, 2006;Lorenz et al, 2009;Canales et al, 2011). In this study, we modified the RNA extraction method from Chang and colleagues (1993) combined with genome grinding/beading strategy to homogenize small amounts of root tissue.…”