cell line: HCT116 cell type: colorectal carcinoma cells genotype: control time point: 16h
Treatment protocol
See Cepeda et al. The ubiquitin ligase SCFFBXO28 regulates Myc-driven transcription through non-proteolytic ubiquitylation and is required for cell proliferation.
Growth protocol
See Cepeda et al. The ubiquitin ligase SCFFBXO28 regulates Myc-driven transcription through non-proteolytic ubiquitylation and is required for cell proliferation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions.
Label
Cy3
Label protocol
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, SuperScript 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, re-apply 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. *50x 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.
See Cepeda et al. The ubiquitin ligase SCFFBXO28 regulates Myc-driven transcription through non-proteolytic ubiquitylation and is required for cell proliferation.
Growth protocol
See Cepeda et al. The ubiquitin ligase SCFFBXO28 regulates Myc-driven transcription through non-proteolytic ubiquitylation and is required for cell proliferation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions.
Label
Cy5
Label protocol
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, SuperScript 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, re-apply 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. *50x 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.
Hybridization protocol
Slide preparation: Slides were pre-hybridised at 42°C for 45 min in the GeneTac hybridisation station. The pre-hybridisation mix consisted of 5x 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).
Scan protocol
Slides were scanned at 10-µm resolution immediately after washing. The photomultiplier tube setting was kept at 100 for all scans. Obtained images were rotated and the mirror image created using the provided software.
Description
Control 16h vs. knockdown 16h.
Data processing
Raw data files were imported into R using the KTH package for microarray analysis. Spots that were either saturated, flagged or had a foreground signal within two standard deviations from that spot's background were removed from further analysis. Individual channel data was converted into MA-format (M=log2 [Cy5/Cy3] ratio, A=log intensity) and within-slide print-tip normalisation was carried out. After manual evaluation of the distrubtion of M- and A-values across arrays, no between-slide normalisation was performed.
