cell line: MCF7 cell type: breast cancer cells genotype/variation: tet-off (tetracycline-repressed) MCF-7-SNAI1 conditional expression of snai1: Induced time point: 4h
Treatment protocol
The tet-off (tetracycline-repressed) MCF-7-SNAI1 cell lines were established. SNAI1 expression in MCF7 stably transfected cells was induced by removing tetracyclin from culture media according to the procedure recommended by BD biosciences Clontech Company (Protocol PT13001-1). To carry out the transcriptional analysis in temporal manner after SNAI1 induction in MCF7-SNAI1 cells, induction of cell was performed at each time point studied. See Vetter and Le Béchec et al. (2009) for more information.
Growth protocol
Cells were grown at 37°C, under 5% CO2 atmosphere. Non-induced MCF7-Tet-Off SNAI1 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum, L-Glutamine, penicillin and streptomycin, G418 (100ug/ml), hygromycin (200ug/ml) and tetracycline (1,5ug/ml). For SNAI1 induction tetracycline was removed from the culture media.
Extracted molecule
total RNA
Extraction protocol
Total RNAs were extracted using Trizol as recommended by manufacturer (Invitrogen).
Label
Cy3
Label protocol
After RNA hybridization, tag-conjugating Cy3 and Cy5 dyes were circulated through the microfluidic chip for dye staining.
cell line: MCF7 cell type: breast cancer cells genotype/variation: tet-off (tetracycline-repressed) MCF-7-SNAI1 conditional expression of snai1: Not induced time point: 4h
Treatment protocol
The tet-off (tetracycline-repressed) MCF-7-SNAI1 cell lines were established. SNAI1 expression in MCF7 stably transfected cells was induced by removing tetracyclin from culture media according to the procedure recommended by BD biosciences Clontech Company (Protocol PT13001-1). To carry out the transcriptional analysis in temporal manner after SNAI1 induction in MCF7-SNAI1 cells, induction of cell was performed at each time point studied. See Vetter and Le Béchec et al. (2009) for more information.
Growth protocol
Cells were grown at 37°C, under 5% CO2 atmosphere. Non-induced MCF7-Tet-Off SNAI1 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum, L-Glutamine, penicillin and streptomycin, G418 (100ug/ml), hygromycin (200ug/ml) and tetracycline (1,5ug/ml). For SNAI1 induction tetracycline was removed from the culture media.
Extracted molecule
total RNA
Extraction protocol
Total RNAs were extracted using Trizol as recommended by manufacturer (Invitrogen).
Label
Cy5
Label protocol
After RNA hybridization, tag-conjugating Cy3 and Cy5 dyes were circulated through the microfluidic chip for dye staining.
Hybridization protocol
The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments. hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies) (Gao et al., 2004; Zhu et al., 2007). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. hybridization used 100 μL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2hPO4, 6 mM EDTA, ph 6.8) containing 25% formamide at 34 °C. Gao, X., Gulari, E., and Zhou, X. (2004) In situ synthesis of oligonucleotide microarrays. Biopolymers 73, 579-596 Zhu, Q., hong, A., Sheng, N., Zhang, X., Jun, K.-Y., Srivannavit, O., Gulari, E., Gao, X., and Zhou, X. (2007) Microfluidic biochip for nucleic acid and protein analysis. in Methods Mol. Biol. Ed. Rampal, J. B. 382:287-312.
Scan protocol
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
Data processing
Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression) (Bolstad et al., 2003). For two color experiments, the ratio of the two sets of detected signals (log2 transformed, balanced) and p-values of the t-test were calculated; differentially detected signals were those with less than 0.01 p-values. Bolstad, B. M., Irizarry, R. A., Astrandand, M., Speed, T. P. (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinfo. 19, 185-193.
