tissue: whole seedlings genetic background: Col-0 genetic variation: transposon gene knock out treatment: DMSO control time: 4hr
Growth protocol
Seed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 ug/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 umol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
Qiagen RNAeasy kit protocol
Label
biotin
Label protocol
Labelled using protocol described in manual "Affymetrix GeneChip Expression Analysis Technical Manual", One-Cycle target labelling
Hybridization protocol
Biotin Labelled cRNA using "Affymetrix GeneChip Hybridization, Wash and Stain Kit", according to guidelines set out by Affymetrix, Santa Clara, CA
Scan protocol
Scanned according to guidelines set out by Affymetrix, Santa Clara, CA.
Description
MS agar plates containing sterilised seed were placed in the dark at 4C for three days. Seed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more. To spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension. Growth Room Agar 0.7% Agar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 ug/ml immediately before pouring into 10 cm square plates. ~200 seeds/plate 20oC 50% Murashige & Skoog basal salt mixture 1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 ug/ml hygromycin B added after sterilisation
Data processing
Raw data from the microarrays was normalized at probe-level using MAS5 algorithm. The detection calls (present, marginal, absent) for each probe set was obtained using the GCOS system
