Three different life stages were sampled: a) seedlings, which had grown just above the soil line, also served as reference tissue; b) leaf shoots from plants with four leaves; and c) leaf shoots from plants with six leaves.
Growth protocol
The plants were grown in a controlled environment chamber set for 25°C, 50 ± 10% relative humidity and 16 hr light/8 hr dark. Lighting was provided by a combination of florescent and incandescent bulbs.
Extracted molecule
total RNA
Extraction protocol
Tissue was ground in a mortar and pestle using liquid nitrogen and RNA was extracted from the powdered tissue using Trizol (Invitrogen, Carlsbad, CA). Extracted RNA was treated with DNase (Qiagen, Valencia, CA) and purified with the RNeasy MinElute Cleanup Kit (Qiagen).
Label
Cy3
Label protocol
Total RNA was converted to amino allyl modified cDNA followed by chemical coupling of Cy5 or Cy3 (GE Healthcare Biosciences, Piscataway, NJ) using the FairPlay III microarray labeling kit (Stratagene, La Jolla, CA). Five different mRNA spikes (SpotReport® Alien® mRNA spikes 1, 3, 5, 7, and 10, Stratagene) were added at varying concentrations to the labeling reaction for scanner normalization. Labeled cDNA was quantified with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and checked for uniform labeling by gel electrophoresis (Liu and Slininger 2007).
Three different life stages were sampled: a) seedlings, which had grown just above the soil line, also served as reference tissue; b) leaf shoots from plants with four leaves; and c) leaf shoots from plants with six leaves.
Growth protocol
The plants were grown in a controlled environment chamber set for 25°C, 50 ± 10% relative humidity and 16 hr light/8 hr dark. Lighting was provided by a combination of florescent and incandescent bulbs.
Extracted molecule
total RNA
Extraction protocol
Tissue was ground in a mortar and pestle using liquid nitrogen and RNA was extracted from the powdered tissue using Trizol (Invitrogen, Carlsbad, CA). Extracted RNA was treated with DNase (Qiagen, Valencia, CA) and purified with the RNeasy MinElute Cleanup Kit (Qiagen).
Label
Cy5
Label protocol
Total RNA was converted to amino allyl modified cDNA followed by chemical coupling of Cy5 or Cy3 (GE Healthcare Biosciences, Piscataway, NJ) using the FairPlay III microarray labeling kit (Stratagene, La Jolla, CA). Five different mRNA spikes (SpotReport® Alien® mRNA spikes 1, 3, 5, 7, and 10, Stratagene) were added at varying concentrations to the labeling reaction for scanner normalization. Labeled cDNA was quantified with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and checked for uniform labeling by gel electrophoresis (Liu and Slininger 2007).
Hybridization protocol
40 picomoles of Cy5 labeled reference cDNA and 40 pmol of Cy3 labeled seedling, 4-leaf, or 6-leaf cDNA was completely evaporated in a speedvac and suspended in 38 μL of sterile water and 2 μL of poly DNA (20 μg/μL). Pre-hybridization of the microarray slide and hybridization of probes to the slide in a sealed hybridization chamber were carried out according to an established protocol (Hedge et al. 2000) except for an additional high stringency wash at room temperature.
Scan protocol
Microarray hybridizations were scanned on a GenePix 4000B scanner (Molecular Devices, Inc., Sunnyvale, CA). Full resolution scanning was analyzed and the scatter plot was used to check for the signal of the mRNA spikes. When necessary, the photomultiplier tube levels were adjusted, and the slide scanned again until the controls had equal signal intensity for both channels.
Data processing
Mean background subtracted, and ratio (Cy3/Cy5) representing test/reference for each microarray chip. Each of the Cy3 labeled seedling, 4-leaf shoot, or 6-leaf shoot treatments were divided by the reference sample labeled Cy5 for each chip.
