Arabidopsis thaliana ecotype Ws (AtHD1/AtHD1, +/+), AtHD1/athd1-t1 (+/–), and athd1-t1/athd1-t1 (–/–) plants were produced.
Growth protocol
The plants were grown in a growth chamber under growth conditions of 22°/18° (day/night) and 14 hr of illumination per day. RNA were isolated from tissues collected from a pool of 32 plants in each line. Rosette leaves were collected at the prebolting stage (~3 weeks), while flower buds were harvested after the first flower bloomed.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the Trizol reagent (Invitrogen, San Diego). The mRNA was isolated from 500 µg of total RNA with the FastTrack 2.0 mRNA isolation kit (Invitrogen).
Label
Cy5
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.
Arabidopsis thaliana ecotype Ws (AtHD1/AtHD1, +/+), AtHD1/athd1-t1 (+/–), and athd1-t1/athd1-t1 (–/–) plants were produced.
Growth protocol
The plants were grown in a growth chamber under growth conditions of 22°/18° (day/night) and 14 hr of illumination per day. RNA were isolated from tissues collected from a pool of 32 plants in each line. Rosette leaves were collected at the prebolting stage (~3 weeks), while flower buds were harvested after the first flower bloomed.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the Trizol reagent (Invitrogen, San Diego). The mRNA was isolated from 500 µg of total RNA with the FastTrack 2.0 mRNA isolation kit (Invitrogen).
Label
Cy3
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
7-37-ws_cy5,athd1_cy3 Biological replicate 2 of 4.
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.
