9 protocols
AccessionType
hybridization
Slide preparation: Slides were pre-hybridised at 42°C for 45 min in the GeneTac hybridisation station. The pre-hybridisation mix consisted of 5 x SSC, 1% BSA (Sigma-Aldrich), 0.1% SDS, 40 µg poly(dA) (Sigma-Aldrich) and 20 µg tRNA (Sigma-Aldrich). Target preparation: Purified targets were pooled for each hybridisation. Hybridisation mixture was added to a final hybridisation composition of 5x SSC, 25% formamide, 0.1% SDS, 40 µg poly(dA) and 20 µg tRNA. The mixture was denaturated at 95 °C for 2 minutes and stored on ice. Hybridisation: Hybridisation in a total volume of 110 µl was carried out for 16-18 hours at 42 °C using continuous agitation. Wash: Hybridised slides were washed for five minutes with 2x SSC and 0.1% SDS at 42°C, followed by 0.1x SSC and 0.1% SDS at room temperature (five minutes) and finally by three one-minute washes with 0.1x SSC at room temperature. Washed slides were immediately dried using a slide centrifuge (Telechem).
labeling
RNA priming: to 20 ug total RNA add 10 ug 20(T)VN primer (MWG Biotech AG), adjust total volume to 18.4 µl, 70C 10 min , ice > 2 min, Prepare 1x RT-mixture (all reagents from Invitrogen): 5x RT-buff 6 µl, 50x aa-dNTP* 0.6 µl, DTT (0.1M) 3 µl, Supersscript II (200 U/µl) 2 µl, cDNA synthesis: To primed RNA, add 11.6 µl RT-mix, add 42C 1 h 45 min, RNA degradation: add 3 µl 200 mM EDTA, add 4.5 µl 1 M NaOH, 70 C 15 min, Neutralisation: add 3 µl 1 M HCl, Cleanup 1: add 70 µl H20 + 500 µl PB, transfer to MinElute column (Qiagen), 13000 rpm 60 s, reapply flow-though, 13000 rpm 1 min, discard flow-through, add 600 µl 80% EtOH, 13000 rpm 60 s, repeat EtOH wash, spin column dry, 13000 rpm 60 s, transfer column to new microtube, add 10 µl 0.1 M NaCO3 pH 9.0, incubate 2 min, 13000 rpm 60 s, add 10 µl 0.1 M NaCO3 pH 9.0, 13000 rpm 60 s, transfer elution to aliquot of Cy5-dye (Amersham)**, mix with pipette, briefly centrifuge, incubate 1.5 h in dark at room temperature, add 4.5 µl 1 M hydroxylamine Cleanup 2 add 60 µl H20 to cDNA, add 500 µl Buffer PB, apply to MinElute column, 13000 rpm 1 min, reapply flow-though, 13000 rpm 1 min, discard flow-through, add 650 mL Buffer PE, 13000 rpm 1 min, dscard flow-through, repeat wash, spin column dry, 13000 rpm 60 s, transfer to fresh microtube, add 10 µl Buffer EB to center of filter, 2 minutes at RT, 13000 rpm 1 min, repeat elution with another 10 µl EB, ------------------------------------------- *50 x aadNTP (4:1 aadUTP:dTTP): 10 µl each 100 mM dATP, dGTP, dCTP (Pharmacia), 8 µl 100 mM aminoallyl-dUTP (Sigma, #A0410), 2 µl 100 mM dTTP, **Aliquot Cy3 dye: dissolve 1 tube in 50 µl DMSO, add 5 µl to 8 tubes. Speed vac dry, store tubes in a dessicator in fridge.
labeling
RNA priming: to 20 ug total RNA add 10 ug 20(T)VN primer (MWG Biotech AG), adjust total volume to 18.4 µl, 70C 10 min , ice > 2 min, Prepare 1x RT-mixture (all reagents from Invitrogen): 5x RT-buff 6 µl, 50x aa-dNTP* 0.6 µl, DTT (0.1M) 3 µl, Supersscript II (200 U/µl) 2 µl, cDNA synthesis: To primed RNA, add 11.6 µl RT-mix, add 42C 1 h 45 min, RNA degradation: add 3 µl 200 mM EDTA, add 4.5 µl 1 M NaOH, 70 C 15 min, Neutralisation: add 3 µl 1 M HCl, Cleanup 1: add 70 µl H20 + 500 µl PB, transfer to MinElute column (Qiagen), 13000 rpm 60 s, reapply flow-though, 13000 rpm 1 min, discard flow-through, add 600 µl 80% EtOH, 13000 rpm 60 s, repeat EtOH wash, spin column dry, 13000 rpm 60 s, transfer column to new microtube, add 10 µl 0.1 M NaCO3 pH 9.0, incubate 2 min, 13000 rpm 60 s, add 10 µl 0.1 M NaCO3 pH 9.0, 13000 rpm 60 s, transfer elution to aliquot of Cy3-dye (Amersham)**, mix with pipette, briefly centrifuge, incubate 1.5 h in dark at room temperature, add 4.5 µl 1 M hydroxylamine Cleanup 2 add 60 µl H20 to cDNA, add 500 µl Buffer PB, apply to MinElute column, 13000 rpm 1 min, reapply flow-though, 13000 rpm 1 min, discard flow-through, add 650 mL Buffer PE, 13000 rpm 1 min, dscard flow-through, repeat wash, spin column dry, 13000 rpm 60 s, transfer to fresh microtube, add 10 µl Buffer EB to center of filter, 2 minutes at RT, 13000 rpm 1 min, repeat elution with another 10 µl EB, ------------------------------------------- *50 x aadNTP (4:1 aadUTP:dTTP): 10 µl each 100 mM dATP, dGTP, dCTP (Pharmacia), 8 µl 100 mM aminoallyl-dUTP (Sigma, #A0410), 2 µl 100 mM dTTP, **Aliquot Cy3 dye: dissolve 1 tube in 50 µl DMSO, add 5 µl to 8 tubes. Speed vac dry, store tubes in a dessicator in fridge.
pool
Each time-point in the study constitutes of three independently grown and treated seedling samples pooled before grounding and RNA extraction.
grow
Arabidopsis thaliana seeds (Col-0) were germinated and grown in 0.5x MS (Duchefa) liquid medium supplemented with 0.5% sucrose at 22C (24 h photoperiod with 16 h of light at 75 mE m-2 sec-1 PAR).
nucleic_acid_extraction
Extraction: Frozen seedlings were ground and total RNA extracted using RNAeasy kit as instructed by the manufacturer (Qiagen GmbH, Hilden, Germany). Sterile RNase-free water was used for elution. No amplification was carried out. Quality control: The RNA Nano kit and the Bioanalyzer instrument (Agilent Technologies) were used to verify the integrity of the obtained RNA. Quantification: NanoDrop® ND-1000 UV-Vis Spectrophotometer (Nanodrop Technologies) was used to quantify the amount of RNA in each sample. Triplicate measurements were carried out and the average of these used. Storage: In small aliquots in a -80 C freezer.
specified_biomaterial_action
Arabidopsis thaliana seeds (Col-0) were germinated and grown in 0.5x MS (Duchefa) liquid medium supplemented with 0.5% sucrose at 22 C 24 h photoperiod with 16 h of light at 75 mE m-2 sec-1 PAR). Treatment for 0, 30, 120 or 240 min with 1 µM indole-3-acetic acid was initiated after 10 days (2 hours after dawn). After treatment seedlings were washed once with 0.5% sucrose for 5 min and immediately frozen in liquid nitrogen and stored at -70 C.
bioassay_data_transformation
Spots corresponding to the intergenic regions were selected and the median of these control foreground (Fg) intensities was computed for each slide. Fg intensity measurements below background (Bg) threshold (i.e. Fg < Bg + 2 x Bg standard deviation) were replaced by the corresponding slide median Fg intergenic control value to reduce the impact of background variability in the statistical analysis of differential expression. The leveled data was Loess normalized by computing the Loess regression from the MA plots, separately for each print-tip and array (Yang, Y.H et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. (2002) 30: e15).
image_acquisition
Slides were scanned at 10-µm resolution immediately after washing. The photo multiplier tube setting was kept at 100 for all scans. Obtained images were rotated and the mirror image created using the provided software.