differentiation stage: two-day postconfluent cells in growth medium cell type: Multipotent Adipose-Derived Stem (hMADS) cells
Treatment protocol
Transient transfection of hMADS cells (when cells had reached confluence, designated day –2) with miRNA mimics (purchased from Dharmacon; miRIDIAN Mimic C-300504-07, hsa-miR-29a, and as non-targeting control miRIDIAN microRNA Mimic Negative Control #1 #CN-001000-01-20) was performed using HiPerFect transfection Reagent (QIAGEN #301707). Briefly, Medium I was changed 1 h before transfection (12 ml per 100 mm dish). The final oligonucleotide concentration was 5.4 nM. Therefore, previously established stock solutions of oligonucleotides (20 µm in sterile PBS, stored at -80°C) were thawed on ice. Subsequently, transfection mixtures were generatedby mixing 3.7 uL oligonucleotide stock solution, 1230 uL DMEM (without any additives) and 74.2 uL HiPerFect transfection reagent (for one 100 mm dish). The reactions were incubated at room temperature for 10 min and finally added dropwise onto the cells while gently swaying the dish. Adipocyte differentiation was induced two days later (designated day 0) by changing the medium to Medium II, consisting of DMEM(1 g/L Glc, Lonza Lonza #BE12-707F)/ Ham’s F12 (Lonza # BE12-615F) (50:50), 2mM L-Gln (Invitrogen/ Gibco #25030-024), 10mM HEPES (Invitrogen/ Gibco #15630-056), 100 µg/mL Normocin (InVivogen #ant-nr-2), 10 nM human insulin (Sigma #I9278), 10 µg/mL apo-transferrin (Sigma #T2252), 0.2 nM T3 (Sigma #T6397), 100 nM rosiglitazone (Cayman Chemicals / BRL49653), 100 µM IBMX (Sigma #I7018) and 1 µm Dexamethasone (Sigma #D4902). 12 ml Medium II were used for 1 100mm dish.
Growth protocol
For experiments, hMADS cells (Rodriguez et al., 2005, PMID: 15867092) were used between passage (P) 19 and P25. Medium for proliferation of hMADS cells was termed Medium I and consisted of (DMEM 1 g/l Glc (Lonza #BE12-707F), 2mM L-Gln (Invitrogen/ Gibco #25030-024), 10mM HEPES (Invitrogen/ Gibco #15630-056), 100ug/ml Normocin (InVivogen #ant-nr-2), 10% FBS (Lonza, #14-801F, Lot.no. 9SB031; or Pan-Biotech, #P30-3300, Lot.no.250330), supplemented with 2.5 ng/mL human Fibroblast Growth Factor 2 (hFGF2, Sigma #F0291). In this medium, hMADS cells had population doubling times of 48-96 h (depending on the passage number, i.e. the replicative age of cells). At densities in the range of 50–80% of optical confluence, passaging of cells was performed at passaging ratios between 1:2 and 1:5, depending on the actual population doubling time. Cells were cultivated in 100 mm dishes (Greiner Bio-One #664160), with 10 ml Medium I per dish, at 37°C in a humidified atmosphere with 5% CO2. For passaging, old medium was aspirated and cells were washed with Phosphate Buffered Saline (PBS, Invitrogen / Gibco #10010015) before incubation with 0.05% Trypsin with EDTA in PBS (Invitrogen / Gibco #15400054; 1mL per 100 mm dish) for 3–5 min at 37°C. Subsequently, Trypsin was inactivated by addition of Medium I (prewarmed to 37°C), and the cell suspension was transferred to new culture dishes according to the desired passaging ratio.
Extracted molecule
total RNA
Extraction protocol
Two days after transfection (= day 0), as well as at day 3 after induction of adipocyte differentiation, cells were harvested with TRIzol reagent (lifetechnologies, #15596-026). Total RNA was isolated according to the manufacturer's instructions and dissolved in RNase-free H2O.
Label
Cy3,Cy5
Label protocol
Concentration of total RNA samples was determined by UV/VIS photospetrometry on a NanoDrop-1000 device, and aliquots of 15 µg total RNA in 16.5 µL RNase-free H2O were prepared. For reverse transcription (RT), 2 µL random primers (3 mg/ml, lifetechnologies # 48190-011) were added to the RNA sample, followed by incubation at 70°C for 10 min and snap-freezing on ice for 30 sec. Subsequently, the following agents were added to complete the RT reactions: 0.6 µL aminoallyl-dNTP mix (25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15mM dTTP (all lifetechnologies #10297-018), 10mM aminoallyl-dUTP (Sigma #A0410)), 6 µL 5x first strand buffer, 3 µL 0.1 M DTT, 2 µL SuperScript II Reverse Transcriptase (all lifetechnologies #18064-014). After mixing, RT reactions were incubated at 42°C for ~18 h. RNA was then hydrolyzed by adding 10 µL 1 M NaOH and 10 µL 0.5 M EDTA, followed by incubation at 65°C for 15 min. Subsequently, pH was neutralized by adding 10 µL 1 M HCl. To remove unincorporated aminoallyl-dUTP, reactions were purified based on the QIAquick PCR purification kit (QIAGEN #28106). Briefly, 300 µL of buffer PB were added to each sample before transfer to a QIAquick column and centrifugation (13000 x rcf, 1 min). Flowthrough was discarded and the cDNA (captured in the column matrix) was washed twice by addition of 750 µL phosphate wash buffer (5 mM potassium phosphate (prepared from 1M potassium phosphate buffer, pH 8.5), 80% EtOH) and subsequent centrifugation and discarding of flowthrough as above. Finally, the column matrix was dried by additional centrifugation for 1 min at maximum speed, and elution of purified cDNA was performed with 60 µL 4 mM potassium phosphate (diluted from 1M potassium phosphate buffer, pH 8.5). Samples were dried completely in a SpeedVac for 45 min and then resuspended in 4.5 µL of 0.1 M Na2CO3 buffer. To label the samples with fluorescent dyes, 4.5 µL of Cy5 or Cy3 NHS-ester (GE healthcare #PA25001 and #PA23001, one aliquot dissolved in 73 µL DMSO and stored at -80°C) were added and the samples were mixed and incubated at room temperature in the dark for 1 h. To remove uncoupled NHS-ester dyes, labeled cDNA samples were purified using the QIAquick PCR purification kit (QIAGEN #28106) according to the manufacturer’s instructions. After elution of the purified labeled cDNA in 60 µL Buffer EB, samples were again completely dried in a SpeedVac for 45 min. Subsequently, samples were resuspended in 24 µL hybridization buffer (50% N,N-Dimethylformamide (Sigma #D4551), 5xSSC, 0.1% SDS) and 1 µL human COT-1 DNA (lifetechnologies #15279-011, 20µg/µL, prepared according to the manufacturer’s instructions) as well as 1 µL poly(A)-DNA (20µg/µL, lifetechnologies primer service) were added to block nonspecific hybridization.
differentiation: two-day postconfluent cells in growth medium cell type: Multipotent Adipose-Derived Stem (hMADS) cells
Treatment protocol
Transient transfection of hMADS cells (when cells had reached confluence, designated day –2) with miRNA mimics (purchased from Dharmacon; miRIDIAN Mimic C-300504-07, hsa-miR-29a, and as non-targeting control miRIDIAN microRNA Mimic Negative Control #1 #CN-001000-01-20) was performed using HiPerFect transfection Reagent (QIAGEN #301707). Briefly, Medium I was changed 1 h before transfection (12 ml per 100 mm dish). The final oligonucleotide concentration was 5.4 nM. Therefore, previously established stock solutions of oligonucleotides (20 µm in sterile PBS, stored at -80°C) were thawed on ice. Subsequently, transfection mixtures were generatedby mixing 3.7 uL oligonucleotide stock solution, 1230 uL DMEM (without any additives) and 74.2 uL HiPerFect transfection reagent (for one 100 mm dish). The reactions were incubated at room temperature for 10 min and finally added dropwise onto the cells while gently swaying the dish. Adipocyte differentiation was induced two days later (designated day 0) by changing the medium to Medium II, consisting of DMEM(1 g/L Glc, Lonza Lonza #BE12-707F)/ Ham’s F12 (Lonza # BE12-615F) (50:50), 2mM L-Gln (Invitrogen/ Gibco #25030-024), 10mM HEPES (Invitrogen/ Gibco #15630-056), 100 µg/mL Normocin (InVivogen #ant-nr-2), 10 nM human insulin (Sigma #I9278), 10 µg/mL apo-transferrin (Sigma #T2252), 0.2 nM T3 (Sigma #T6397), 100 nM rosiglitazone (Cayman Chemicals / BRL49653), 100 µM IBMX (Sigma #I7018) and 1 µm Dexamethasone (Sigma #D4902). 12 ml Medium II were used for 1 100mm dish.
Growth protocol
For experiments, hMADS cells (Rodriguez et al., 2005, PMID: 15867092) were used between passage (P) 19 and P25. Medium for proliferation of hMADS cells was termed Medium I and consisted of (DMEM 1 g/l Glc (Lonza #BE12-707F), 2mM L-Gln (Invitrogen/ Gibco #25030-024), 10mM HEPES (Invitrogen/ Gibco #15630-056), 100ug/ml Normocin (InVivogen #ant-nr-2), 10% FBS (Lonza, #14-801F, Lot.no. 9SB031; or Pan-Biotech, #P30-3300, Lot.no.250330), supplemented with 2.5 ng/mL human Fibroblast Growth Factor 2 (hFGF2, Sigma #F0291). In this medium, hMADS cells had population doubling times of 48-96 h (depending on the passage number, i.e. the replicative age of cells). At densities in the range of 50–80% of optical confluence, passaging of cells was performed at passaging ratios between 1:2 and 1:5, depending on the actual population doubling time. Cells were cultivated in 100 mm dishes (Greiner Bio-One #664160), with 10 ml Medium I per dish, at 37°C in a humidified atmosphere with 5% CO2. For passaging, old medium was aspirated and cells were washed with Phosphate Buffered Saline (PBS, Invitrogen / Gibco #10010015) before incubation with 0.05% Trypsin with EDTA in PBS (Invitrogen / Gibco #15400054; 1mL per 100 mm dish) for 3–5 min at 37°C. Subsequently, Trypsin was inactivated by addition of Medium I (prewarmed to 37°C), and the cell suspension was transferred to new culture dishes according to the desired passaging ratio.
Extracted molecule
total RNA
Extraction protocol
Two days after transfection (= day 0), as well as at day 3 after induction of adipocyte differentiation, cells were harvested with TRIzol reagent (lifetechnologies, #15596-026). Total RNA was isolated according to the manufacturer's instructions and dissolved in RNase-free H2O.
Label
Cy5,Cy3
Label protocol
Concentration of total RNA samples was determined by UV/VIS photospetrometry on a NanoDrop-1000 device, and aliquots of 15 µg total RNA in 16.5 µL RNase-free H2O were prepared. For reverse transcription (RT), 2 µL random primers (3 mg/ml, lifetechnologies # 48190-011) were added to the RNA sample, followed by incubation at 70°C for 10 min and snap-freezing on ice for 30 sec. Subsequently, the following agents were added to complete the RT reactions: 0.6 µL aminoallyl-dNTP mix (25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15mM dTTP (all lifetechnologies #10297-018), 10mM aminoallyl-dUTP (Sigma #A0410)), 6 µL 5x first strand buffer, 3 µL 0.1 M DTT, 2 µL SuperScript II Reverse Transcriptase (all lifetechnologies #18064-014). After mixing, RT reactions were incubated at 42°C for ~18 h. RNA was then hydrolyzed by adding 10 µL 1 M NaOH and 10 µL 0.5 M EDTA, followed by incubation at 65°C for 15 min. Subsequently, pH was neutralized by adding 10 µL 1 M HCl. To remove unincorporated aminoallyl-dUTP, reactions were purified based on the QIAquick PCR purification kit (QIAGEN #28106). Briefly, 300 µL of buffer PB were added to each sample before transfer to a QIAquick column and centrifugation (13000 x rcf, 1 min). Flowthrough was discarded and the cDNA (captured in the column matrix) was washed twice by addition of 750 µL phosphate wash buffer (5 mM potassium phosphate (prepared from 1M potassium phosphate buffer, pH 8.5), 80% EtOH) and subsequent centrifugation and discarding of flowthrough as above. Finally, the column matrix was dried by additional centrifugation for 1 min at maximum speed, and elution of purified cDNA was performed with 60 µL 4 mM potassium phosphate (diluted from 1M potassium phosphate buffer, pH 8.5). Samples were dried completely in a SpeedVac for 45 min and then resuspended in 4.5 µL of 0.1 M Na2CO3 buffer. To label the samples with fluorescent dyes, 4.5 µL of Cy5 or Cy3 NHS-ester (GE healthcare #PA25001 and #PA23001, one aliquot dissolved in 73 µL DMSO and stored at -80°C) were added and the samples were mixed and incubated at room temperature in the dark for 1 h. To remove uncoupled NHS-ester dyes, labeled cDNA samples were purified using the QIAquick PCR purification kit (QIAGEN #28106) according to the manufacturer’s instructions. After elution of the purified labeled cDNA in 60 µL Buffer EB, samples were again completely dried in a SpeedVac for 45 min. Subsequently, samples were resuspended in 24 µL hybridization buffer (50% N,N-Dimethylformamide (Sigma #D4551), 5xSSC, 0.1% SDS) and 1 µL human COT-1 DNA (lifetechnologies #15279-011, 20µg/µL, prepared according to the manufacturer’s instructions) as well as 1 µL poly(A)-DNA (20µg/µL, lifetechnologies primer service) were added to block nonspecific hybridization.
Hybridization protocol
Shortly before hybridization, microarrays were washed in 0.2% SDS (3 times, 2 min each) and ddH2O (4 times, 1 min each), then incubated in prehybridization buffer (5x SSC, 0.1% SDS, 1% BSA) at 42°C for 45 min, again washed with ddH2O (five times, 30 sec each), finally dipped in isopropanol, and dried by immediate centrifugation. Immediately before applying the labeled cDNA samples to the microarray, the samples were heated at 95°C for 3 min, snap cooled on ice for 30 sec and centrifuged for 1 min at maximum speed. The sample was pipetted onto the microarray surface, and a rectangular coverslip was gradually lowered, distributing the sample across the spotted area of the microarray. Hybridization was performed by incubation at 42°C for 16-20 h. Subsequently, microarrays were submerged in wash buffer I (2x SSC, 0.1% SDS, preheated to 42°C) for 2 min, leading to separation of the microarray and the coverslip. Three washing steps (each 5 min at room temperature with gentle rotation) were performed using wash buffer I (2x SSC, 0.1% SDS, preheated to 30°C), wash buffer II (1x SSC), and wash buffer III (0.5xSSC). Finally, the microarray was dipped into ddH2O and dried by immediate centrifugation.
Scan protocol
Scanning of microarrays was performed with a GenePix 4000B microarray scanner (Axon Instruments) at 10 µm resolution using GenePix Pro 4.1 software (Axon Instruments).
Description
Biological replicate 2, cells harvested 2 days after transfection
Data processing
GenePix Pro 4.1 was used to filter out spots of suboptimal quality prior to further analysis. High intensity spots were filtered out if more than 20% of spot pixels were saturated in either of the two channels. Inhomogenity of spots was estimated by comparing (for either of the two channels) the median of pixel intensities to the mean; if the difference was greater than 20%, the spot was considered as inhomogenous and thus filtered out. Low intensity spots were defined by two distinct approaches: (i) if the sum of the medians/means of the pixel intensities in both channels was lower than 700, or (ii) if not more than 55 percent of the pixels within a spot had intensities higher than the intensity of the surrounding background plus one standard deviation of the background pixels. Subsequently, the filtered raw data was background corrected (by estimation and subtraction of local background), normalized by global mean and dye-swap pairs using ArrayNorm (PMID: 15073026). Finally, features detecting the same transcript were combined by averaging and the data was exported as text-file containing log2 transformed ratios of gene expression.
