Raw data results were processed using GenePix Pro 4.1 software (Axon Instruments) to filter out low intensity, saturated and inhomogenous spots. Next, the data was background corrected, normalized by global mean and dye-swap pairs using ArrayNorm (Pieler et al, 2004), and then exported as text-file containing log2 transformed ratios of gene expression. Subsequently, only transcripts present in at least 3 biological replicates were selected and further filtered for transcripts specified by NCBI RefSeq ID. Last, features with identical RefSeq IDs (i.e., detecting the same transcript) were merged by averaging of expression values within each biological replicate.
TITLE: HUMAN OLIGO CHIP (HOC) PROBE HYBRIDIZATION SOP-NO: MET015_03 BASED ON: TIGR SOP# M005 PAGE: 1 of 6 REWIEV PERIOD: biannual VALID FROM: 01/03/2004 originated: the Dipl.-Chem. Dr. M. Scheideler date approved by: the Dipl.-Ing. Dr.techn., Univ.- Prof. Z. Trajanoski date Contents I ....... PURPOSE II ...... SCOPE III ..... MATERIAL IV ….. REAGENT PREPARATION V …… PROCEDURE I. PURPOSE This protocol describes the hybridization of a Cy labeled cDNA probe (mix of Cy3 and Cy5) onto epoxy coated slide spotted with 50mer oligonucleotides. II. SCOPE This procedural format is currently utilized by Human Oligo Chip microarray projects under the supervision of Zlatko Trajanoski within the Bioinformatics Group, Institute for Genomics and Bioinformatics, Graz University of Technology, Graz Austria. III. MATERIAL 3.1 20x Saline-Sodium Citrate (SSC) (Sigma; Cat # S-6639) 3.2 10% Sodium Dodecyl Sulfate (SDS) (Life Technologies; Cat # 15553-035) 3.3 Bovine Serum Albumin (BSA) (Sigma; Cat # A-9418) 3.4 Formamide, redistilled (Life Technologies; Cat # 15515-081) 3.5 Isopropanol (Fisher Scientific; Cat # A451-1) 3.6 Coplin jar (VWR; Cat # 25457-200) 3.7 Human COT1-DNA (Life Technologies; Cat # 15279-011) 3.8 Mouse COT1-DNA (Life Technologies; Cat # 18440-016) 3.9 Poly(A)-DNA (Pharmacia; Cat # 27-7836-01) 3.10 Microscope Cover Glass (Fisher Scientific; Cat # 12-545J) 3.11 Human Oligo Chips produced by spotting 50mer oligonucleotides (MWG, Ebersberg) onto epoxy coated glass slides (Quantifoil) at the Bioinformatics Group, Biophysics Dept., Institute of Biomedical Engineering, Graz University of Technology, Graz Austria 3.12 Hybridization chamber (Corning Costar; Cat #2551) 3.13 1 L .22 µm CA (cellulose acetate) Filter System (Corning; Cat #430517) 3.14 Pressurized air duster (Fellowes; Cat # 99790) or clean in-house pressurized air source IV. REAGENT PREPARATION 4.1 Prehybridization Buffer 4.1.1 To make 500 ml Prehybridization Buffer combine: Volume Percentage Volume Starting Material End Concentration 25 % 125 ml 20x SSC 5x SSC 1 % 5 ml 10 % SDS 0.1 % SDS 5 g BSA 1 % BSA 74 % 370 ml MilliQ Water 4.2 1x Hybridization Buffer 4.2.1 To make 1 mL Hybridization Buffer combine: Volume Percentage Volume Starting Material End Concentration 50 % 500 µl 99.5 % Formamide 50 % Formamide 25 % 250 µl 20x SSC 5x SSC 1 % 10 µl 10 % SDS 0.1 % SDS 24 % 240 µl MilliQ Water 4.3 Wash Buffer I (low stringency buffer like MWG protocol) 4.3.1 To make 1 L Wash Buffer I combine: Volume Percentage Volume Starting Material End Concentration 10 % 100 ml 20x SSC 2x SSC 1 % 10 ml 10 % SDS 0.1 % SDS 89 % 890 ml MilliQ Water 4.4 Wash Buffer II (low stringency buffer like MWG protocol) 4.4.1 To make 1 L Wash Buffer II combine: Volume Percentage Volume Starting Material End Concentration 5 % 50 ml 20x SSC 1x SSC 95 % 950 ml MilliQ Water 4.5 Wash Buffer III (high stringency buffer like MWG protocol) 4.5.1 To make 1 L Wash Buffer III combine: Volume Percentage Volume Starting Material End Concentration 2.5 % 25 ml 20x SSC 0.5x SSC 97.5 % 975 ml MilliQ Water V. PROCEDURE 5.1 Capture Probe Fixation 5.1.1 Epoxy coated slides (Quantifoil) spotted with 50mer oligonucleotides in 3x SSC, 1.5 M Betaine are baked at 42 °C for 8 hours at 50% rel. humidity. 5.2 Slide Washing 5.2.1 Prepare 150 ml 0.2% SDS washing buffer per microarray and fill it in 3 Coplin jars. 5.2.2 Wash the printed slide(s) which will be used for the hybridization three times in a Coplin jar containing 0.2% SDS washing buffer by incubating at room temperature for 2 minutes on a shaker. 5.2.3 Wash the printed slide(s) four times in a Coplin jar containing ddH2O by incubating at room temperature for 1 minute on a shaker. 5.2.4 Dry the slide by immediate centrifugation at 1500 rpm for 2 minutes. 5.3 Prehybridizaton 5.3.1 Prepare prehybridization buffer (5x SSC, 0.1% SDS, 1% BSA) and sterilize by filtration using a CA filter. Preheat at 42 °C for ~30 minutes before use. 5.3.2 Place the printed slide(s) which will be used for the hybridization in a Coplin jar containing preheated prehybridization buffer and incubate at 42 °C for 45 minutes. 5.3.3 Washing Slides - Fill five Coplin jars with MilliQ water and another with isopropanol. - With forceps carefully grasp slide by the labeled end and vertically dip slide into the first Coplin jar (water) so that the slide is completely submerged. Dip slide five times. - Dip the slide again in the water five times but only submerging the slide enough to wash the printed array itself. - Using the same technique dip slide into the following Coplin jars (water). - Finally dip the slide into the sixth Coplin jar (isopropanol) submerging the slide completely. Note: Replace each water wash after every five slides. 5.3.4 Dry slides by immediate centrifugation at 1500 rpm for 2 minutes Note: - Note the general appearance of the slide. Streaking or mottling on the slide surface indicates further washing is necessary. - Repeat the water/water/isopropanol wash cycle as necessary to clean the slide. Blow dry between each cycle. - When working with an oligonucleotide array one can denature the spotted oligos by submerging the slide in a 95 °C MilliQ water bath for 3 minutes just after prehybridization and before adding the probe. 5.3.5 Use slides immediately following prehybridization to ensure optimal hybridization efficiency. 5.4 Hybridization 5.4.1 Prepare 1x hybridization buffer (50% formamide, 5x SSC, and 0.1% SDS). 5.4.2 Prepare Poly(A)-DNA by dissolving stock Poly(A)-DNA in a neutral buffer (i.e. 10 mM Tris, pH 7) to a final concentration of 20 µg/µL. 5.4.3 Prepare COT1-DNA (stock conc.1µg/µL) by ethanol precipitation: - Add 2 to 3 volumes of ethanol and 0.1 volumes of 3 M Sodium Acetate (NaOAc) to the stock tube. - Mix well and place on dry ice for 20-30 minutes or in –20 °C freezer overnight. - Centrifuge for 20-30 minutes in a cold room microfuge at maximum angular velocity. - Remove supernatant and allow excess ethanol to dry off. - Dissolve precipitated COT1 in a neutral buffer (i.e. 10 mM Tris, pH 7) to the final concentration of 20 µg/µL. 5.4.4 Resuspend labeled probe (Cy3/Cy5 probe mixture: see SOP-Met014) in 24 µL of 1x hybridization buffer. Note: Expose Cy labeled probe to light as little as possible during the hybridization process. 5.4.5 To block nonspecific hybridization add: COT1-DNA (20 µg/µL)……. 1µL Poly(A)-DNA (20µg/µL)…... 1µL Note: The COT1–DNA is organism specific: add mouse COT1 to labeled mouse probes and human COT1-DNA to labeled human probes. 5.4.6 To denature, heat the probe mixture at 95 °C for 3 minutes and snap cool on ice for 30 sec. 5.4.7 Centrifuge the probe mixture at maximum angular velocity for 1 minute. Keep at room temperature and use immediately. 5.4.8 To Apply Labeled Probe Mixture - Place a prehybridized microarray slide (array side up) between the guide teeth in the bottom half of a hybridization chamber. - Pipette the labeled probe mixture (~26 µL) to the slide surface near one end of the array print area keeping bubbles to a minimum. - Take a 22mm x 60mm microscope glass coverslip, dust it with compressed air, and grasp one end with forceps. - Holding the coverslip over the array print area, lower the end nearest the pool of cDNA probe until solution wicks to the surface of the coverslip. - Gradually lower the opposite end of the coverslip (held by the forceps) onto the slide. The solution may take a minute or two to wick across the entire length of the slide. - After probe has wicked across the slide carefully adjust the coverslip’s position with the tip of the forceps so that there is an even margin between the edge of the coverslip and the edge of the slide. - Work any large bubbles toward the edge by gently tapping the coverslip surface; small bubbles will absolve themselves during hybridization. 5.4.9 To the small wells at each end of the chamber add 10 µL of water (20µL total), cover, and seal the chamber. 5.4.10 Wrap the chamber in foil (light-tight) and incubate in a 42 °C water bath for 16-20 hours. To ensure chamber remains level and does not float to the surface place a small weight upon it. Note: Do not flip the hybridization chamber upside down during hybridization; this may cause the coverslip to shift from the slide and adversely affect the hybridization. 5.5 Washing Slides after hybridization 5.5.1 Prepare a low stringency wash buffer I (~500mL) containing 2x SSC and 0.1% SDS 5.5.2 Prepare a low stringency wash buffer II (~500mL) containing 1x SSC 5.5.3 Prepare a high-stringency wash buffer III (~500mL) containing 0.5x SSC. 5.5.4 Preheat all wash buffers (I-III) to 30 °C before usage. Fill one Coplin jar with wash buffer I and preheat to 42 °C before usage. 5.5.5 After the incubation remove foil and unseal hybridization chamber. Remove the slide from the chamber, taking care not to disturb the coverslip. 5.5.6 To remove coverslip submerge slide in a the Coplin jar containing low stringency wash buffer I (preheated to 42 °C) for 2 minutes. With time the coverslip will slide free of the slide surface. Note: Once the slide has been hybridized it should be exposed to light as little as possible. Therefore, all staining dishes should be covered with foil to make them light tight. 5.5.7 After the coverslip is removed place slide in a staining dish containing low stringency wash buffer I (2x SSC and 0.1% SDS, preheated to 30 °C) and agitate for 5 minutes at room temperature. 5.5.8 Wash the slide in a staining dish with low stringency wash buffer II (1x SSC, preheated to 30 °C) by agitating for 5 minutes at room temperature. 5.5.9 Wash the slide in high-stringency wash buffer III (0.5x SSC, preheated to 30 °C) agitating for 5 minutes at room temperature. 5.5.10 Dip the slide shortly in a copling jar filled with MilliQ water. 5.5.11 Finally dry the slides by immediate centrifugation at 1500 rpm for 2 minutes using the same technique as step 4.2.4. 5.5.12 Place slides in a light tight slide box until they can be scanned, preferably as soon as possible. 4 Institute for Genomics and Bioinformatics Graz University of Technology Erzherzog-Johann-University HUMAN OLIGO CHIP (HOC) PROBE HYBRIDIZATION SOP-NO: MET015_03 Page 2 of 7 4 \\Majestix\genome\Documents\GLP\newSOPs\SOP MET015_03_HOC_µA Probe Hybridization.doc Institute for Genomics and Bioinformatics Graz University of Technology Erzherzog-Johann-University Standard Operating Procedure \\Majestix\genome\Documents\GLP\newSOPs\SOP MET015_03_HOC_µA Probe Hybridization.doc
TITLE: AMINOALLYL LABELING OF RNA FOR HUMAN OLIGO CHIPS (HOC) SOP-NO: MET014_01 BASED ON: TIGR SOP# M004 PAGE: 1 of 6 REWIEV PERIOD: biannual VALID FROM: 01/01/2004 originated: the Dipl.-Chem. Dr. M. Scheideler date approved by: the Dipl.-Ing. Dr.techn., Univ.- Prof. Z. Trajanoski date Contents I ....... PURPOSE II ...... SCOPE III ..... PROCEDURE I. PURPOSE This protocol describes the labeling of eukaryotic RNA with aminoallyl labeled nucleotides via first strand cDNA synthesis followed by a coupling of the aminoallyl groups to either Cyanine 3 or 5 (Cy 3/Cy5) fluorescent molecules. II. SCOPE This procedural format is currently utilized by Human Oligo Chip microarray projects under the supervision of Zlatko Trajanoski within the Bioinformatics Group, Biophysics Dept., Institute of Biomedical Engineering, Graz University of Technology, Graz Austria. III. MATERIAL 3.1 5-(3-aminoallyl)-2’deoxyuridine-5’-triphosphate (AA-dUTP) (Sigma; Cat # A0410) 3.2 100 mM dNTP Set PCR grade (Life Technologies; Cat # 10297-018) 3.3 Random Hexamer primers (3mg/mL) (Life Technologies; Cat # 48190-011) 3.4 SuperScript II RT (200U/µL) (Life Technologies; Cat # 18064-014) 3.5 Cy-3 ester (AmershamPharmacia; Cat # PA23001) 3.6 Cy-5 ester (AmershamPharmacia; Cat # PA25001) 3.7 QIAquick PCR Purification Kit (Qiagen; Cat # 28106) 3.8 RNeasy® Mini Kit (Qiagen; Cat # 74106) IV. REAGENT PREPARATION 4.1 Phosphate Buffers 4.1.1 Prepare 2 solutions: 1M K2HPO4 and 1M KH2PO4 4.1.2 To make a 1 M Phosphate buffer (KPO4, pH 8.5-8.7) combine: 1 M K2HPO4……..9.5 ml 1 M KH2PO4……..0.5 mL 4.1.3 For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 1 M KPO4 pH 8.5…. 0.5 mL MilliQ water………... 15.25 mL 95% ethanol………. 84.25 mL Note: Wash buffer will be slightly cloudy. 4.1.4 Phosphate elution buffer is made by diluting 1 M KPO4, pH 8.5 to 4 mM with MilliQ water. 4.2 Aminoallyl dUTP 4.2.1 For a final concentration of 100 mM add 19.1 µL of 0.1 M KPO4 buffer (pH 7.5) to a stock vial containing 1 mg of aa-dUTP. Gently vortex to mix and transfer the aa-dUTP solution into a new microfuge tube. Store at –20 °C. 4.2.2 Measure the concentration of the aa-dUTP solution by diluting an aliquot 1:5000 in 0.1 M KPO4 (pH 7.5) and measuring the OD289. (Stock concentration in mM = OD289 x 704) 4.3 Labeling Mix (50x) with 2:3 aa-dUTP: dTTP ratio 4.3.1 Mix the following reagents: Final concentration dATP (100 mM)………5µL…... (25 mM) dCTP (100 mM)………5µL…... (25 mM) dGTP (100 mM)………5µL…... (25 mM) dTTP (100 mM)………3µL…… (15 mM) aa-dUTP (100 mM)…......2µL…… (10 mM) Total: 20µL 4.3.2 Store unused solution at –20 °C. 4.4 Sodium Carbonate Buffer (Na2CO3): 1 M, pH 9.0 4.4.1 Dissolve 10.8 g Na2CO3 in 80 mL of MilliQ water and adjust pH to 9.0 with 12 N HCl; bring volume up to 100 mL with MilliQ water. 4.4.2 To make a 0.1 M solution for the dye coupling reaction dilute 1:10 with water. Note: Carbonate buffer changes composition over time; make it fresh every couple of weeks to a month. 4.5 Cy-dye esters 4.5.1 Cy3-ester and Cy5-ester are provided as a dried product in 5 tubes. Resuspend a tube of dye ester in 73 µL of DMSO before use. 4.5.2 Wrap all reaction tubes with foil and keep covered as much as possible in order to prevent photobleaching of the dyes. Note: Dye esters must either be used immediately or aliquotted and stored at –80 °C. Any introduced water to the dye esters will result in a lower coupling efficiency due to the hydrolysis of the dye esters. Since DMSO is hygroscopic (absorbs water from the atmosphere) store it well sealed in desiccant. V. PROCEDURE 5.1 Aminoallyl Labeling 5.1.1 To 20 µg of total RNA (or 2 µg poly(A+) RNA) which has been DNase I-treated and Qiagen RNeasy purified, add 2 µL Random Hexamer primers (3mg/mL) and bring the final volume up to 18.5 µL with RNase-free water. 5.1.2 Mix well and incubate at 70 °C for 10 minutes. 5.1.3 Snap-freeze in dry ice/ethanol bath for 30 seconds, centrifuge briefly at >10,000 rpm and continue at room temperature. 5.1.4 Add: 5x First Strand buffer………….. 6 µL 0.1 M DTT………………………….. 3 µL 50x aminoallyl-dNTP mix……… 0.6 µL SuperScript II RT (200 U/µL)… 2 µL 5.1.5 Mix and incubate at 42 °C for 3 hours to overnight. 5.1.6 To hydrolyze RNA, add: 1 M NaOH 10 µL 0.5 M EDTA 10 µL mix and incubate at 65 °C for 15 minutes. 5.1.7 Add 10 µL of 1 M HCl to neutralize pH. (Alternatively, one can add 25 µL 1 M HEPES pH 7.0 or 25 µL 1 M Tris pH 7.4) 5.2 Reaction Purification I: Removal of unincorporated aa-dUTP and free amines (use the Qiagen method) Qiagen Cleanup Method: Note: This purification protocol is modified from the Qiagen QIAquick PCR purification kit protocol. The phosphate wash and elution buffers (prepared in 4.1.3 & 4.1.4) are substituted for the Qiagen supplied buffers because the Qiagen buffers contain free amines which compete with the Cy dye coupling reaction. 5.2.1 Mix cDNA reaction with 300 µL (5x reaction volume) buffer PB (Qiagen supplied) and transfer to QIAquick column. 5.2.2 Place the column in a 2 ml collection tube (Qiagen supplied) and centrifuge at ~13,000 rpm for 1 minute. Empty collection tube. 5.2.3 To wash, add 750 µL phosphate wash buffer to the column and centrifuge at ~13,000 rpm for 1 minute. 5.2.4 Empty the collection tube and repeat the wash and centrifugation step (5.2.3). 5.2.5 Empty the collection tube and centrifuge column an additional 1 minute at maximum speed. 5.2.6 Transfer column to a new 1.5 mL microfuge tube and carefully add 30 µL phosphate elution buffer (see 4.1.4) to the center of the column membrane. 5.2.7 Incubate for 1 minute at room temperature. 5.2.8 Elute by centrifugation at ~13,000 rpm for 1 minute. 5.2.9 Elute a second time into the same tube by repeating steps 5.2.6- 5.2.8. The final elution volume should be ~60 µL. 5.2.10 Dry sample in a speed vac with 45 minutes run time and 30 minutes medium heating time. 5.3 Coupling aa-cDNA to Cy Dye Ester 5.3.1 Resuspend aminoallyl-labeled cDNA in 4.5 µL 0.1 M sodium carbonate buffer (Na2CO3), pH 9.0. Note: Carbonate buffer changes composition over time so make sure you make it fresh every couple of weeks to a month. 5.3.2 Add 4.5 µL of the appropriate NHS-ester Cy dye (prepared in DMSO: see 4.5) Note: To prevent photobleaching of the Cy dyes wrap all reaction tubes in foil and keep them sequestered from light as much as possible. 5.3.3 Incubate the reaction for 1 hour in the dark at room temperature. 5.4 Reaction Purification II: Removal of uncoupled dye (Qiagen PCR Purification Kit) 5.4.1 To the reaction add 35 µL 100 mM NaOAc pH 5.2. 5.4.2 Add 250 µL (5x reaction volume) Buffer PB (Qiagen supplied). 5.4.3 Place a QIAquick spin column in a 2 mL collection tube (Qiagen supplied), apply the sample to the column, and centrifuge at ~13,000 for 1 minute. Empty collection tube. 5.4.4 To wash, add 750 µL Buffer PE (Qiagen supplied) to the column and centrifuge at ~13,000 for 1 minute. Note: Make sure Buffer PE has added ethanol before using (see label for correct volume). 5.4.5 Empty collection tube and centrifuge column for an additional 1 minute at maximum speed. The column bottom should have the colour of the incorporated dye. 5.4.6 Place column in a clean 1.5 mL microfuge tube and carefully add 30 µL Buffer EB (Qiagen supplied) to the center of the column membrane. 5.4.7 Incubate for 1 minute at room temperature. 5.4.8 Elute by centrifugation at ~13,000 rpm for 1 minute. 5.4.9 Elute a second time into the same tube by repeating steps 5.4.6- 5.4.8. The final elution volume should be ~60 µL. The solution should have the colour of the incorporated dye. Note: This protocol is modified from the Qiagen QIAquick Spin Handbook (04/2000, pg. 18). 5.5 Analysis of Labeling Reaction (optional) 5.5.1 Use a 50 µL Beckman quartz MicroCuvette to analyze the entire und diluted sample in a spectrophotometer. 5.5.2 Wash the cuvette with water and blow dry with compressed air duster. 5.5.3 Pipette sample into cuvette and place cuvette in spectrophotometer. 5.5.4 For each sample measure absorbance at 260 nm and either 550 nm for Cy3 or 650 nm for Cy5, as appropriate. 5.5.5 Pipette sample from cuvette back into the original sample tube. 5.5.6 For each sample calculate the total picomoles of cDNA synthesized using: pmol nucleotides = [OD260 * volume (µL) * 37 ng/µL * 1000 pg/ng] 324.5 pg/pmol Note: 1 OD260 = 37 ng/µL for cDNA; 324.5 pg/pmol average molecular weight of a dNTP) 5.5.7 For each sample calculate the total picomoles of dye incorporation (Cy3 or Cy5 accordingly) using: pmol Cy3 = OD550 * volume (µL) 0.15 pmol Cy5 = OD650 * volume (µL) 0.25 nucleotides/dye ratio = pmol cDNA pmol Cy dye Note: >200 pmol of dye incorporation per sample and a ratio of less than 50 nucleotides/dye molecules is optimal for hybridizations (see Microarray Cookbook II) 5.6 Drying Slides 5.6.1 Dry the Cy3/Cy5 probe mixture to completion in a speed vac and continue with SOP: Met015 for the hybridization of the probe to a microarray slide. 4 Institute for Genomics and Bioinformatics Graz University of Technology Erzherzog-Johann-University AMINOALLYL LABELING OF RNA FOR HUMAN OLIGO CHIPS (HOC) SOP-NO: MET014_01 Page 6 of 6 4 \\Majestix\genome\Documents\GLP\newSOPs\SOP MET014_01_HOC_aa Labeling of RNA for µAs.doc Institute for Genomics and Bioinformatics Graz University of Technology Erzherzog-Johann-University Standard Operating Procedure \\Majestix\genome\Documents\GLP\newSOPs\SOP MET014_01_HOC_aa Labeling of RNA for µAs.doc
Total RNA of cells was obtained using TRIzol reagent (Invitrogen, Cat.no.15596-026) according to the manufacturer's protocol.
Transient transfection of hMADS cells (at day –2) with oligonucleotides (siRNAs, miRNA mimics or anti-miRNA antisense oligonucleotides, see below for details) was performed using HiPerFect transfection Reagent (QIAGEN #301707). Briefly, Medium I (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 (for hMADS-2 cells: Pan-Biotech #P30-3300, Lot.no. P250330; for hMADS-3 cells: Lonza, #14-801F, Lot.no. 9SB031) was changed 1 h before transfection (1ml per well of a 12-well plate (Greiner Bio-One #665180), 12ml per 100 mm dish (Greiner Bio-One #664160)). Standard transfections had a final oligonucleotide concentration of 5 nM. Therefore, stock solutions of oligonucleotides (20 µm in sterile PBS, stored at -80°C) were thawed on ice and diluted to 2 µm working solutions using sterile, nuclease-free H2O. Subsequently, transfection mixtures were generated as follows: A) 12-well-format: 3 uL oligonucleotide working solution, 97 uL DMEM (without any additives) and 6 uL HiPerFect Transfection reagent per well of a 12-well plate; B) 100 mm-dish-format: 30uL oligonucleotide working solution, 1161 uL DMEM (without any additives) and 72 uL HiPerFect Transfection reagent per 100 mm dish. The reactions were mixed and incubated at room temperature for 10 min and finally added dropwise onto the cells while gently swaying the plate/dish. Unless cells were harvested, medium was changed after two days. In experiments with an oligonucleotide concentration higher than 5 nM, dilution of the stock solutions was adjusted appropriately, and the volume of HiPerFect transfection reagent was scaled as recommended by the manufacturer. Commonly used oligonucleotides: A) miRIDIAN Mimic hsa-miR-26a: Dharmacon #C-300499-05-0005 B) miRIDIAN Mimic Negative Control #1: Dharmacon #CN-001000-01-20
For experiments, hMADS cells originally established from 2 different donors were used. hMADS-2 cells were isolated from the pubic fat pad of a 5 year old male donor; hMADS-3 cells were isolated from the prepubic fat pad of a 4 months old male donor (Rodriguez et al., 2005, PMID: 15867092). Cells between passage (P) 17 and P32 were used. 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 (for hMADS-2 cells: Pan-Biotech #P30-3300, Lot.no. P250330; for hMADS-3 cells: Lonza, #14-801F, Lot.no. 9SB031), 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. 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 the respective growth medium (prewarmed to 37°C), and the cell suspension was transferred to new culture dishes according to the desired passaging ratio. The usual volume of medium was 10mL per 100 mm cell culture dish. Cells were incubated at 37°C in a humidified atmosphere with 5% CO2. For some experiments, hMADS cells were seeded in 12-well plates (Greiner Bio-One #665180). Therefore, the cells were trypsinized as described above, followed by a determination of cell concentration using a hemocytometer (Neubauer #T728.1). Subsequently, cells were seeded at densities between 5000 and 7500 cells per cm2. The final volume of cell suspension (in Medium I supplemented by hFGF2) was 1 ml per well of a 12-well plate. Usually, hMADS cells reached optical confluence (designated d-2) 4 to 6 days after seeding. For adipocyte differentiation, hMADS cells were cultivated in Medium I (without hFGF2) for 2 additional days after reaching optical confluence (i.e. until d0). Subsequently, adipocyte differentiation was induced by 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), 860 µM 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). At d2 or d3, the medium was changed to Medium II without IBMX and Dex (designated “Medium III”). For differentiation into brown/brite/beige (i.e. UCP1-positive) adipocytes, cells were kept in this medium until d14–d17, whereas for white adipocyte differentiation, rosiglitazone was omitted from the medium from d9 on. Generally, media were changed every two to three days and the media volumes were 1mL per well of a 12-well plate and 12 ml per 100 mm dish.