Genotype: B73xH99 F1 hybrid Tissue: Immature ear Stage: V15 Origin: bulk of 10 individuals
Growth protocol
Immature ears were harvested from plants cultivated in open field (2003), at 12 p.m. along 3 different days
Extracted molecule
polyA RNA
Extraction protocol
After harvesting, plant material was immediately frozen after removing silks and ear apexes and stored at - 80 C. Tissues were ground in liquid nitrogen using mortars and pestles. Total RNA was isolated using the TRizol protocol (Invitrogen, Carlsbad, CA), as indicated by the manufacturer (except for 5 min extra time centrifugation in TRizol reagent), including a second step in chloroform for lowering protein contamination. Poly(A+) RNA was purified from 1 mg of total RNA derived from a minimum of three independent extractions using mRNA Purification Kit (Amersham Bioscience, Little Chalfont, UK). Both total and poly(A+) RNA were tested for quality by electrophoresis on 1.5% agarose gel and quantified by absorbance at 260 nm.
Label
Cy3
Label protocol
1 ug of purified poly(A+) RNA was independently retrotranscribed using 400 U of SuperScript II RNase H- Reverse Transcriptase (Invitrogen, Carlsbad, CA) and 2 ug Oligo(dT)23 Anchored (Sigma-Aldrich, St. Louis, MO) as primer, in 30 uL final volume (2 hours, 42 C). cDNA probes were labeled by direct incorporation of Cy3/Cy5 modified dCTP 0.3 mM (Amersham Bioscience, Little Chalfont, UK); dATP, dGTP and dTTP 0.5 mM each, dCTP 0.2 mM. Reaction was stopped adding 1.5 uL EDTA (0.5 M – pH 8.0) and 3.75 uL NaOH (1 M) (10 min, 65 C) and then neutralized with 0.75 uL HCl (5 M) and 9 uL Tris HCl (1 M – pH 6.9). Probe was purified with Nucleo Spin Extract kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), protocol #4.2 with double wash in NT3 buffer.
Immature ears were harvested from 10 individuals cultivated in open field (2003), at 12 p.m. along 3 different days
Extracted molecule
polyA RNA
Extraction protocol
After harvesting, plant material was immediately frozen after removing silks and ear apexes and stored at - 80 C. Tissues were ground in liquid nitrogen using mortars and pestles. Total RNA was isolated using the TRizol protocol (Invitrogen, Carlsbad, CA), as indicated by the manufacturer (except for 5 min extra time centrifugation in TRizol reagent), including a second step in chloroform for lowering protein contamination. Poly(A+) RNA was purified from 1 mg of total RNA derived from a minimum of three independent extractions using mRNA Purification Kit (Amersham Bioscience, Little Chalfont, UK). Both total and poly(A+) RNA were tested for quality by electrophoresis on 1.5% agarose gel and quantified by absorbance at 260 nm.
Label
Cy5
Label protocol
1 ug of purified poly(A+) RNA was independently retrotranscribed using 400 U of SuperScript II RNase H- Reverse Transcriptase (Invitrogen, Carlsbad, CA) and 2 ug Oligo(dT)23 Anchored (Sigma-Aldrich, St. Louis, MO) as primer, in 30 uL final volume (2 hours, 42 C). cDNA probes were labeled by direct incorporation of Cy3/Cy5 modified dCTP 0.3 mM (Amersham Bioscience, Little Chalfont, UK); dATP, dGTP and dTTP 0.5 mM each, dCTP 0.2 mM. Reaction was stopped adding 1.5 uL EDTA (0.5 M – pH 8.0) and 3.75 uL NaOH (1 M) (10 min, 65 C) and then neutralized with 0.75 uL HCl (5 M) and 9 uL Tris HCl (1 M – pH 6.9). Probe was purified with Nucleo Spin Extract kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), protocol #4.2 with double wash in NT3 buffer.
Hybridization protocol
After adding 12 ug of Polydeoxyadenylic Acid (Amersham Bioscience, Little Chalfont, UK) the probe was lyophilized in SpeedVac SVC-100 H (Savant Instruments/E-C Apparatus, Holbrook, NY) and then resuspended in 29 uL Array Hyb Low Temp Hybridization Buffer (Sigma-Aldrich, St. Louis, MO) and 2 uL salmon sperm DNA (20 ug/uL). Slides were re-hydrated 7 min in water-saturated atmosphere and briefly dried on heating plate (3-4 sec); spotted cDNA were cross-linked to the silane-glass support applying twice 65 mJ/cm2 UV light (254 nm; Stratalinker 2400 UV cross-linker, Stratagene, La Jolla, CA). After rinsing 2 min in SDS at R.T., spotted cDNA were denatured by immersion of slides in mQ water for 2 min at 95 C. Unspecific binding sites were blocked for 40 min at 65 C in 1% BSA, 3.5X SSC, 0.2% SDS. Slides were rinsed at R.T. in mQ water 50 times in each of four trays, then in iso-propanol (10 immersions), and finally air-dried and stored in a clean box until hybridization. After denaturation (2 min, 98 C in mQ water) probe was hybridized on microarray slides o. n. at 50 C in a dark hybridization chamber (CMT-Hybridization Chamber, Corning Inc., Corning, NY). Slides were then washed in SDS 1% solutions at increasing stringency (SSC concentration 2X, 1X, 0.1X; 10 min each, 65 C), then in SSC 0.1X (5 min, 65 C, twice), air-dried and stored in the dark at room temperature until image acquisition.
Scan protocol
Microarray images for Cy3 and Cy5 channels were acquired using ScanArray software on SA4000 Scanner (v3.1, Packard BioScience, Wellesley, MA), setting laser power to 90% and auto-adjusting photo-multiplicator gain to the maximum sub-saturating value for each channel. Signal and background intensities and spot parameters were quantified by QuantArray (v3.0, Packard BioScience, Wellesley, MA).
Description
Slide number 606.01.04.029. For a detailed description of the experiment see: Pea G, Ferron S, Gianfranceschi L, Krajewski P, Pe' ME (2007) Gene expression non-additivity in immature ears of a heterotic F1 maize hybrid. Plant Science (DOI:10.1016/j.plantsci.2007.09.005)
Data processing
Records corresponding to single bad-quality spots were manually removed. Intensity data were imported into GeneSpring (Agilent Technologies, Palo Alto, CA), where local background subtraction as well as per-chip LOWESS and 50th percentile within-array normalization functions were performed on log-two transformed data. Base two logarithms of hybrid/inbred expression ratios for each EST, calculated from the average over three replicate spots within the slide, were exported from GeneSpring to perform all subsequent analyses.
