|
| Status |
Public on Nov 20, 2008 |
| Title |
Gene expression difference between A. suecica and their progenitor species |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Leaf tissue mRNAs from Arabidopsis suecica
|
| Organism |
Arabidopsis suecica |
| Characteristics |
Natural allopolyploid species formed by interspecific hybridization between extant A.thaliana and A.arenosa species.
|
| Biomaterial provider |
Z. Jeffrey Chen, The University of Texas at Austin
|
| Treatment protocol |
All plants were grown in a growth chamber at 22 degree and under 16 hour of light per day.
|
| Growth protocol |
All plants were grown in a growth chamber at 22 degree and under 16 hour of light per day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies), following the manufacture instruction. mRNAs were isolated from total RNA using a Micro-FastTrack 2.0 mRNA isolation kit (Invitrogen).
|
| Label |
Cy3 and Cy5, dye swap
|
| 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, and then an equal amount of RNA samples was reversely labeled for another hybridization.
|
| |
|
| Channel 2 |
| Source name |
Artificial mix of mRNAs from Leaf tissues of A. thaliana and A. arenosa
|
| Organisms |
Arabidopsis thaliana; Arabidopsis arenosa |
| Characteristics |
A.thaliana and A.arenosa are extant progenitor species of A.suecica
|
| Biomaterial provider |
Z. Jeffrey Chen, The University of Texas at Austin
|
| Treatment protocol |
All plants were grown in a graoth chamber at 22 degree and under 16 hour of light per day.
|
| Growth protocol |
All plants were grown in a graoth chamber at 22 degree and under 16 hour of light per day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies), following the manufacture instruction. mRNAs were isolated from total RNA using a Micro-FastTrack 2.0 mRNA isolation kit (Invitrogen). Equal amount of mRNA from A.thaliana and A.arenosa were combined as a midparent value to detect nonadditive expression in a natural allopolyploid species, A.suecica.
|
| Label |
Cy3 and Cy5, dye swap
|
| 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, and then an equal amount of RNA samples was reversely labeled for another 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 |
8 replicate measurements. Four biological replications were performed and each biological replication was measured by dye-swap experiments. One biological replication was performed using 12 plants per species. Four biological replications were performed with different pools of the plants and extraction. To exclude dye effect, A.suecica and midparent value were reversely labeled and hybridized for individual biological replication.
|
| Data processing |
We applied a linear model to exclude technical variation by arrays and dyes and biological variation by different plant populations. The linear model is
Log (Yigkl) = µ + Gi + Tj + Ak + Dl + (G*T)ij + (G*A)ik + (G*D)il + (G*T*D)ijl + ɛijkl
Where Y represents raw intensity after background level is subtracted; G, T, A, and D are main sources of variation from gene (G), treatment (plant species, T), array (A) and dye (D); i = 1,…, 31818; j = 1, 2; k = 1, 2, 3, 4;l = 1,2; µ represents the overall mean. The interaction terms, G*T, G*A, G*D, and G*T*D mean gene-by-species, gene-by-array, gene-by-dye and gene-by-species-by-dye. ɛijkl represents random error.
To test difference of mRNA expression between A.suecica and mid-parent value (MPV), t-tests were employed for individual genes (pergene-variance).
The null hypothesis, H0: (T)A.suecica + (G*T)i,A.suecica = (T)MPV + (G*T)i,MPV was examined by p-value. Overall type I error rate of multiple testing was controlled to be below 0.05 employing the false discovery rate of Benjamini and Hotchberg.
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| |
|
| Submission date |
Oct 16, 2008 |
| Last update date |
Nov 17, 2008 |
| Contact name |
Misook Ha |
| E-mail(s) |
misook.ha@gmail.com
|
| Phone |
7732795900
|
| Organization name |
National Heart Lung Blood Institute
|
| Lab |
Laboratory of Epigenome Biology
|
| Street address |
NIH
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL7498 |
| Series (2) |
| GSE9513 |
Duplicate genes increase expression diversity in closely related species and allopolyploids |
| GSE13468 |
Gene expression change in natural allotetraploid, A.suecica |
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