7 protocols
Data analysis.Microarray data analysis was performed with free software genArise, developed in the Computing Unit of Cellular Physiology Institute of UNAM (http://www.ifc.unam.mx/genarise/). GenArise carry out a number of transformations: background correction, lowess normalization, intensity filter, replicates analysis and selecting differentially expressed genes. The goal of GenArise is to identify which of the genes show good evidence of being differentially expressed. The software identifies differential expressed genes by calculating an intensity-dependent z-score. Using a sliding window algorithm to calculate the mean and standard deviation within a window surrounding each data point, and define a z-score where z measures the number of standard deviations a data point is from the mean.zi = (Ri û mean(R)) / sd(R)Where zi is the z-score for each element, Ri is the log-ratio for each element, and sd(R) is the standard deviation of the log-ratio. Ratio calculations of significant changes in gene expression derived from globally normalized data are performed by simply computing the ratio of the average of all of the measurements from one condition or sample to another. With this criterion, the elements with a z-score > 2 standard deviations would be the significantly differentially expressed genes (Cheadle et al., 2003).
Labbeling Protocol. 10 ug of total RNA were used for cDNA synthesis incorporating dUTP-Alexa555 or dUTP-Alexa647 employing the First-Strand cDNA labeling kit (Invitrogen). Incorporation of fluorophore was analyzed by using the absorbance at 555 nm for Alexa555 and 650 nm for Alexa647.
(Parameters: Amplification = none, Mass unit = Micro gram)
Equal quantities of labeled cDNA were hybridized using hybridization solution UniHyb (TeleChem International INC), to the 30,000 oligos A. thaliana arrays for 14 h at 42 degrees C.Experimental design: Chips Alexa555 Alexa647AthV3_03_11 WS-3 1 es1-D 1AthV3_03_12 es1-D 1 WS-3 1
(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 10, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 42)
Plants were germinated in soil in a growth chamber at 22 degrees C under long day conditions (8 hrs dark/16 hrs light) and transferred to standard greenhouse conditions.
(Parameters: time unit = seconds, temperature unit = C)
RNA isolation modified from (Verwoerd et al., 1989):RNA extraction buffer: 100 mM Tris pH 9.5 , 1% SDS, 100 mM LiCl, 10 mM EDTA, Phenol pH 7.5, Chloroform, 8 M LiCl; 33.9 g/100 ml70% EtOH. 1) Remove tissue from -70 degrees C freezer to liquid N2. Weight the deeply frozen material quickly and restore in liquid N2. Chill mortar and pestle with liquid N2, add the plant material to the liquid N2 in the mortar, and start grinding the tissue just before the N2 has evaporated. Continue grinding until a very fine powder remains. 2) Fill 50 ml Greiner Blue-cap tube with 5 ml extraction buffer per gram of tissue and add about an equal volume of phenol.3) Quickly transfer the powder with cooled spatula into this mixture and vortex vigorously for about 30 sec. 4) Transfer the blue-cap tube to a 60 degrees C waterbath. Incubate for 5 min and shake once a while. 5) Spin at 3000 rpm for 10 min. 6) Extract supernatant with a ? volume of Chloroform (5 ml). 7) Spin at 3000 rpm for 5 min. 8) Add to the supernatant 1/3 volume of 8 M LiCl (final concentration 2 M LiCl), transfer mixture to eppendorf tubes and incubate O/N at 4 degrees C.9) Spin 10 min. max speed at 4 degrees C.10) Wash pellet with 70% EtOH and spin again for 5 min. 11) Dry the pellet briefly and dissolve pellet in 50 ul water or TE per gram of tissue.(Parameters: Extracted product = total_RNA, Amplification = none)
RNA was extracted from pooled inflorescences of 20 plants containing closed buds only, comparing the empty siliques mutant es1-D (in Ws-3 background) with wild type Arabidopsis plants (Ws-3).
Scanning Protocol. Acquisition and quantification of array images was performed in ScanArray 4000 with its accompanying software ScanArray 4000 from Packard BioChips. All images were captured using 65% PMT gain, 70 to 75% laser power and 10?m resolution at 50% scan rate. For each spot the Alexa555 and Alexa647 density mean value (Dty), background mean value (Bck) and Signal (Sig) cross channel loees normalization value (by subgrids) were calculated with software ArrayPro Analyzer from Media Cibernetics.
(Parameters: Scanning hardware = OTHER: ScanArray 4000 from Packard BioChips, Scanning software = Scanning software)