leaves from two-week seedlings grown under standard conditions in greenhouse (16 h photoperiod and 20-24°C temperature) in pots containing composite soil, reference sample (pooled leaves)
Extracted molecule
total RNA
Label
Cy5
Description
The RNA (800ng) samples were labeled by using SuperScript Indirect RNA Amplification System Kit (Invitrogen, cat# L1016-02) based on the method designed by Eberwine y col. 1992. Following RNA amplification (with the incorporation of UTP aminoallil), labeled product was achieved by incubating with Cy3 or Cy5 esters in alkaline media. Dye-swaps were used to correct for differences in incorporation and fluorescent properties of both dyes, generating a number of 9 slides per experiment (three slides for control and three slides for each treatment) with a total number of 18 slides considering technical replicates. The microarray slides were prehybridized by incubation in 5X SSC, 0.1% SDS, 1% BSA at 42°C. The next day the cover slip was removed and the slide was washed once in 1X SSC, 0.2% SDS prewarmed to 42°C; once in 0.2X SSC, 0.2% SDS at room temperature; and once in 0.1X SSC at room temperature. The washes were conduced with gentle shaking at 100 rpm for 5 minutes. Slides were subjected to low speed centrifugation for 2 min at 500 rpm to dry. The hybridized slides were scanned using a VersArray Chip Reader (BioRad) scanner (two different channels for the two different dyes were used) at three different detector sensitivities. Images analysis and signal quantification were performed using Spotfinder (free open source software) (www.tm4.org/spotfinder.html), quantifying signal intensity for each spot. Then, data integration from multiple scanning processes were achieved
Data processing
Background subtraction was performed before calculating ratios. The elements with either printing or hybridization artifacts were flagged and discarded before analysis. Only spots with an intensity of at least 1.5 times above the local background in both channels were used for subsequent analysis. The extracted data from each slide were then log transformed (using log base two) and normalized using two different methods: 3-D normalization (normalization intensity and space dependant) (Alvarez y col, unpublished data) using R package (University of Auckland) R 1.9.0 version (http://www.r-project.org). Potential artifacts and false positives were eliminated by selecting for further analysis only those clones that exhibited similar expression patterns between the original hybridization and their dye swaps (Yang y col. 2002). A gene expression matrix was generated and its analysis was focused on genes differentially expressed. The whole analysis related to gene expression matrix was performed using software Infostat 2006® (Infostat Group, FCA, Córdoba, Argentina).
