Processing the Loblolly Pine PtGen2 cDNA Microarray

Summary

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.

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 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

1. Mix and briefly spin each kit component before use.
2. Prepare reactions as follows:
   • Xµl DEPC-H

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 BD	352070
15mL Conical Tubes	Other	Falcon BD	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-Aldrich	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 Healthcare	PA25001
Cy-3 Dye	Reagent	GE Healthcare	PA23001
LifterSlips™	Other	Erie Scientific	25X601
HybChambers	Equipment	GeneMachines	JHYB200003