THE INSTITUTE FOR GENOMIC RESEARCH TITLE: MICROARRAY LABELED PROBE HYBRIDIZATION SOP #: M005 REVISION LEVEL: 2 EFFECTIVE DATE: 3/4/02 AUTHOR: Jeremy Hasseman PRIMARY REVIEWERS: Emily Chen, Ivana Yang 1. PURPOSE This protocol describes the hybridization of a Cy labeled cDNA probe (mix of Cy3 and Cy5) onto coated slide spotted with PCR amplified cDNA or 70mer oligonucleotides. 2. SCOPE This procedural format is currently utilized by Human Colon Cancer and Mouse microarray projects under the supervision of John Quackenbush within the Eukaryotic Genomics Dept. 3. MATERIALS 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 ArrayIt SuperAmine Substrates (Telechem International; Cat # SMM- 25) 3.12 Hybridization chamber (Corning Costar; Cat #2551) 3.13 1 L .22 um CA (cellulose acetate) Filter System (Corning; Cat #430517) 3.14 Pressurized air duster (Fellowes; Cat # 99790) or clean in-house pressurized air source 4. PROCEDURE 4.1 UV Cross-linking Slides (using Stratagene UV Stratalinker® 1800) 4.1.1 Aminosilane coated slides (ArrayIt SuperAmine Substrates) spotted with cDNA in 50% DMSO are UV cross-linked at ~120 mJ. 4.1.2 Aminosilane coated slides spotted with 70mer oligos are UV cross-linked at ~450 mJ. 4.2 Prehybridizaton 4.2.1 Prepare prehybridization buffer (5X SSC, 0.1% SDS, 1% BSA) and sterilize by filtration using a CA filter. Preheat at 42oC for ~30 minutes before use. 4.2.2 Place the printed slide(s) which will be used for the hybridization in a Coplin jar containing preheated prehybridization buffer and incubate at 42oC for 45 minutes. 4.2.3 Washing Slides - Fill two 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 four or 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 second Coplin jar (water). - Finally dip slide into the third Coplin jar (isopropanol) submerging the slide completely. Note: Replace each water wash after every five slides. 4.2.4 Drying Slides - Carefully but quickly blow dry the front of the slide with compressed air. Note: To avoid blowing debris from the forceps onto the array, first blow from where the array ends toward the forceps. Then dry the array itself by blowing down the slide away from the forceps. - Finally dry the back of the slide. - 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. Note: When working with a 70mer oligo array one can denature the spotted oligos by submerging the slide in a 95o C MilliQ water bath for 3 minutes just after prehybridization and before adding the probe. 4.2.5 Use slides immediately following prehybridization to ensure optimal hybridization efficiency. 4.3 Hybridization 4.3.1 Prepare 1X hybridization buffer (50% formamide, 5X SSC, and 0.1% SDS). Note: 70mer oligo arrays use a 1X hybridization buffer without formamide and are subsequently hybridized at 65oC. 4.3.2 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 ug/uL. 4.3.3 Prepare COT1-DNA (stock conc.1ug/uL) 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 -20oC 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 20ug/uL. 4.3.4 Resuspend labeled probe (Cy3/Cy5 probe mixture: see SOP-M004) in 24 uL of 1X hybridization buffer. Note: Expose Cy labeled probe to light as little as possible during the hybridization process. 4.3.5 To block nonspecific hybridization add: COT1-DNA (20 ug/uL) ... 1uL Poly(A)-DNA (20ug/uL) ... 1uL Note: The COT1-DNA is organism specific: add mouse COT1 to labeled mouse probes and human COT1-DNA to labeled human probes. 4.3.6 To denature, heat the probe mixture at 95oC for 3 minutes and snap cool on ice for 30 sec. 4.3.7 Centrifuge the probe mixture at maximum angular velocity for 1 minute. Keep at room temperature and use immediately. 4.3.8 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 uL) 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. 4.3.9 To the small wells at each end of the chamber add 10 uL of water (20uL total), cover, and seal the chamber. 4.3.10 Wrap the chamber in foil (light-tight) and incubate in a 42oC 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: 70mer oligo arrays are hybridized without formamide and thus are placed in 65oC water bath for 16-20 hours. 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. 4.3.11 Prepare a low stringency wash buffer (~500mL) containing 1X SSC and 0.2% SDS and a high-stringency wash buffer (~500mL) containing 0.1X SSC and 0.2% SDS. 4.3.12 After the incubation remove foil and unseal hybridization chamber. Remove the slide from the chamber, taking care not to disturb the coverslip. 4.3.13 To remove coverslip submerge slide in a dish containing low stringency wash buffer (preheated to 42oC). With forceps shake the slide gently to loosen the coverslip. 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. 4.3.14 After the coverslip is removed place slide in a staining dish containing lower stringency wash buffer (preheated to 42oC) and agitate for 4 minutes. 4.3.15 Wash the slide in a staining dish with high stringency wash buffer by agitating for 4 minutes at room temperature. 4.3.16 Wash the slide in 0.1X SSC agitating for 4 minutes at room temperature. (alternatively one can wash twice in 0.1X SSC agitating for 2.5 minutes to minimize SDS carryover.) 4.3.17 Finally dip the slide in a Coplin jar filled with water several times and blow dry using the same technique as step 4.1.4. 4.3.18 Place slides in a light tight slide box until they can be scanned, preferably as soon as possible.
Animal handling and RNA preparation at the Medical College of Wisconsin. I. Animal handling at Medical College of Wisconsin == Forty-eight rats are used per study, with eight groups of six rats each. 6 Female parental normoxic 6 Male parental normoxic 6 Female parental hypoxic 6 Male parental hypoxic 6 Female consomic normoxic 6 Male consomic normoxic 6 Female consomic hypoxic 6 Male consomic hypoxic == Each group of six rats is divided into two pools of three animals each, with each pool containing tissues from 3 rats. Pool A Pool B == Four individual tissues from all three animals are pooled together. Heart Kidney Liver Lung pool pool pool pool 48 rats / 3 rats per pool X 4 tissues = 64 pooled samples *Note - In some cases, pools may contain less than 3 rats due to death of animal. == Animals are on a twelve-hour light/dark cycle (light 6AM-6PM). The rats diet is Teklad chow with 0.4% NaCl (order # 3075S, see http://www.teklad.com/index.htm for more information) and water ad libitum. == At 10 weeks of age, animals are subjected to an environmental stressor. One group is moved to hypoxia conditioning chambers, while the second, normoxic, group is left at baseline/standard conditions. The hypoxia chambers maintain fixed rate airflow with 12% oxygen, while the standard oxygen level is 21%. == At 12 weeks, the rats are fasted, water ad libitum, for twelve hours (+/- one hour) before sacrifice. II. Tissue sampling at Medical College of Wisconsin 1) Tissue collection is performed in the necropsy room of MCWs Animal Resource Center (ARC). The following equipment is used in tissue collection setup: Transponder reader Decapicones(R) Labeled 50ml tubes containing RNAlater Labeled 1.5ml tubes for liver DNA sample Dissecting tools Saline Cotton swabs Gauze pads Liquid Nitrogen Guillotine Dissecting board w/ rib spreaders 2) Fasting begins at 10PM. Tissue collection begins at 10AM and is completed within two hours. 3) Three technicians contribute to the tissue collection. One \"clean\" technician organizes the collection process and keeps records, while two harvest tissues. The procedure is done as follows: a) Confirm all animal ID #s with transponder reader and record on tissue harvest pool form b) Place a conscious, non-anesthetized rat into a plastic restraint cone and decapitate, with the head falling directly into liquid nitrogen. Drain blood until muscle activity ceases and then open the rat at the midline. c) Remove and mince heart, right kidney, one liver lobe, and one lung lobe into individual labeled tubes containing 15ml RNAlater(TM), with no pieces larger than 0.5cm in one direction. (A liver sample is also taken for DNA archival at MCW and collected in 1.5ml screw top tube.) d) Remove the transponder from the rat and dispose of the carcass in ARC burn cooler. e) Rinse dissecting tools with sterile saline between animals within a pool. f) Procedure is repeated with tissues from two additional rats being pooled into the same tube. g) Replace saline between pools. 4) Tissues are places in 4°C refrigerator following collection for up to 1 month, then placed @ -20°C for archival. III. RNA extraction at Medical College of Wisconsin ]] Equipment used in RNA extraction: Trizol(TM) reagent Chloroform Isopropanol 75% Ethanol Powergen homogenizer Saw tooth generators (7X195mm and 10X195mm) Centrifuge (capable of 12000Xg) 50ml and 15ml conical tubes Microcentrifuge tubes 1) Remove the tissue from RNAlater and weigh; add 1ml of Trizol reagent(TM) per 100mg of tissue to 50ml conical tube, with a maximum of 500mg of tissue homogenized at once. 2) Homogenize tissue pools in Trizol using a saw tooth generator (7X195mm or 10X195mm) on a Powergen homogenizer. 3) Incubate the homogenate at room temperature for five minutes, and then add 0.2ml of chloroform per 1ml of Trizol used. Shake sample for 15 sec by hand, then incubate at room temperature for 5 minutes. 4) Spin the samples at 10,000Xg, 4-8°C, for 15 minutes. 5) Transfer aqueous layer to new 15ml tube and add 0.5ml Isopropanol per 1ml of Trizol. (An aliquot of RNA in isopropanol, approximately 1.5ml, is archived at MCW at -80°C.) Incubate samples at room temperature for 15 minutes, and then repeat spin. 6) Remove isopropanol and wash pellet, containing RNA, with 75% Ethanol. Air-dry tubes for 10 minutes to evaporate ethanol. 7) Re-suspend the pellet with Diethylpyrocarbonate (DEPC) treated water and warm to 55°C for 10 minutes. Repeat spin (step 4) for five minutes to removed any material not in solution. 8) Place supernatant, containing RNA, in a new microcentrifuge tube. 9) Measure the samples spectrophotometrically at 260/280nm to obtain RNA concentration. 10) Store the samples at -80°C. IV. RNA clean up == The extracted RNA pool is run through Qiagens RNeasy midi kit to remove any remaining genomic DNA or degraded RNA. Kit contains buffers and tubes, user must supply 96-100% Ethanol, ß-Mercaptoethanol, and DNase (optional). All centrifugation steps are done at 3000-5000Xg. The Qiagen RNA cleanup protocol is used with an optional DNase treatment, as follows: 1) Bring sample up to volume with water, as indicated in chart. 2) Add Buffer RLT, according to chart above, and mix. 3) Add 100% Ethanol and shake, and then apply 4ml of sample to RNeasy midi column in 15ml tube. 4) Centrifuge for five minutes at 3000-5000Xg (If sample volume is over 4ml, discard flow through and repeat spin with remaining sample). 5) Optional DNase treatment- Total RNA in sample Add DEPC H20 to Buffer RLT Ethanol <500µg 500µl 2.0ml 1.4ml 500-1000µg 1000µl 4.0ml 2.8ml a) Pipet 2ml Buffer RW1 into column and spin for 5 minutes, then discard flow through. b) Combine 20ul DNase I stock with 140ul Buffer RDD for each sample, and add to column. c) Incubate at room temp for 15 min. d) Add 2ml RW1 and incubate at room temp 5 min, then repeat spin for 5 min. 6) Discard flow-through and add 2.5ml Buffer RPE, and centrifuge for 2 min. Discard flow-through and add another 2.5 ml RPE, and repeat centrifuge 5 min. 7) Place column in new 15ml tube and add 250µl DEPC water, let stand 1 min, centrifuge (as in s 3 min to elute RNA (use only 150µl water if sample is less than 150µg RNA). 8) Repeat elution (can use first elute for increased concentration, but yield will be decreased) 9) The concentration of the samples is measured as before and 1ug of sample is run on a 1% agarose gel to assess quality (Gel images are stored at MCW). 10) Total RNA is stored at -80°C in labeled 2ml tubes until shipment. 11) Samples are then sent to TIGR, by priority overnight shipment on dry ice, for microarray analysis.
Follow manufacturers instructions.