A synthetic allopolyploid line generated by hybridization of Arabidopsis thaliana and Arabidopsis arenosa
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was isolated using Trizol reagent (Invitrogen, San Diego) according to the manufacturer's recommendations. Each RNA sample was quantified by measuring 260/280 ratios using a UV-spectrometer (GeneQuant pro; Amersham Biosciences, Arlington Heights, IL) and by agarose–formaldehyde gel electrophoresis. Total RNA was subjected to mRNA isolation, using a Micro-FastTrack 2.0 mRNA isolation kit (Invitrogen). Equal amounts of mRNA from A. thaliana and A. arenosa were mixed as a midparent value to detect nonadditive gene expression in the allotetraploids
Label
Either Cy3 or Cy5, dye-swap experiments
Label protocol
We used 500 ng of mRNA in each labeling reaction using Cy3- or Cy5-dCTP (Amersham Biosciences). The Cy3-dCTP reaction was mixed with the Cy5-dCTP reaction for one hybridization
Channel 2
Source name
Artificial mix of mRNAs from Leaf tissues of A. thaliana and A. arenosa
Arabidopsis thaliana and Arabidopsis arenosa are progenitor species of the synthetic allopolyploid line, Allo733
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was isolated using Trizol reagent (Invitrogen, San Diego) according to the manufacturer's recommendations. Each RNA sample was quantified by measuring 260/280 ratios using a UV-spectrometer (GeneQuant pro; Amersham Biosciences, Arlington Heights, IL) and by agarose–formaldehyde gel electrophoresis. Total RNA was subjected to mRNA isolation, using a Micro-FastTrack 2.0 mRNA isolation kit (Invitrogen). Equal amounts of mRNA from A. thaliana and A. arenosa were mixed as a midparent value to detect nonadditive gene expression in the allotetraploids
Label
Either Cy3 or Cy5, dye-swap experiments
Label protocol
We used 500 ng of mRNA in each labeling reaction using Cy3- or Cy5-dCTP (Amersham Biosciences). The Cy3-dCTP reaction was mixed with the Cy5-dCTP reaction for one hybridization
Hybridization protocol
Each lyophilized probe was re-suspended in 40 μL of hybridization solution (0.25 M Na2HPO4, 0.25 M NaH2PO4, pH 7.4, and 3.5% SDS, w/v). The solution was heated for 2 min at 95 °C, chilled immediately in ice, and applied directly to the array. After covering the array with a 24 × 40 mm coverslip (Sigma, St Louis, MO), the slide was placed in a microarray hybridization chamber (Corning Incorporated, Corning, NY). Hybridization was performed overnight (16 h) at 60 °C in a hybridization oven. After hybridization, the slides were washed for 2 min in 2× SSC, 0.03% (w/v) SDS, 2 min in 0.2× SSC, and 2 min in 0.05× SSC. Immediately after the last wash, the slides were dried by centrifugation (3 min at 500 r.p.m.).
Scan protocol
Slides were scanned using Genepix 4000B
Description
Total 8 replications ( 4 biological replications and dye-swap experiments were performed for each biological replication).
Data processing
The data were processed using a lowess function to remove nonlinear components and analyzed using a linear model. This linear model was employed to partition variation in the observed data relative to technical and biological variation. Given that each feature is represented once on an array, the linear model is
Yijklm ¼ m1Ai 1Dj 1Tk 1Gl 1AGil 1DGjl 1TGkl 1TDGjkl 1eijklm
where µ represents the overall mean effect; A, D, T, and G represent main fixed effects from the array, dye, treatment (e.g., RNA from two species), and gene, respectively; and i = 1, ..., 8, k = 1, 2, j = 1, 2, and l = 1, ..., 27,648 (including 26,090 70-mer Arabidopsis oligos plus controls). The interaction terms AG, DG, TG, and TDG represent array-by-gene, dye-by-gene, treatment-by-gene, and treatment-by-dye-by-gene interactions, and {varepsilon}ijklm denotes the random error and is used to test for significance of main and interaction effects in the model. Due to confounding and/or aliasing issues involving the array, dye, and treatment terms, not all two-way interactions are included in the model. The model residuals are assumed to be normally distributed with a common variance [i.e., {varepsilon}ijklm iid N(0, {sigma}2)], unless evidence of variance nonconstancy is observed. In such a case, a per gene variance is assumed [i.e., {varepsilon}ijklm independent N(0, {sigma}l2)].
We tested differential expression using significant differences in T + TG terms for a particular gene because we are interested in changes in expression beyond the average treatment effect. The hypotheses reflect whether a gene, g, has undergone differential expression between treatments, t and t' (e.g., A. thaliana and A. arenosa).
A standard t-test statistic is used for this comparison, based on the normality assumption for the residuals. To control for multiple testing errors the false discovery rate (FDR) of BENJAMINI and HOCHBERG (1995) was employed as it provides weak control of the familywise error rate (FWER) and controls the FDR below level {alpha}. The FDR is defined as the expected proportion of incorrect rejections of H0, relative to the total number of rejections. The significance level {alpha} = 0.05 was chosen for these investigations. All analyses of variance models were fit using standard statistical packages
