biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsYonghong Wangtranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-TIGR-7Examination of gene expression associated with asthma induced by treatment of mice with LPS in a controlled environment. Related experiment E-TIGR-8biostudies-arrayexpressHybridization - Title: SOP_M005_3. Description: THE INSTITUTE FOR GENOMIC RESEARCHStandard Operating Procedure TITLE: MICROARRAY LABELED PROBE HYBRIDIZATION PAGE: 1 of 5 SOP #: M005 REVISION LEVEL: 3 EFFECTIVE DATE: 9/11/02 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. 2. SCOPE This procedural format is currently utilized by Human Colon Cancer and Mouse microarray projects under the supervision of John Quackenbush within the Mammalian 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 Corning UltraGAPSä Coated Slides (bar-coded) (Corning; Cat # 40015) 3.12 Hybridization chamber (Corning Costar; Cat #2551) 3.13 1 L .22 mm 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 and Prehybridizaton 4.1.1 Aminosilane coated slides (Corning UltraGAPSä Coated Slides) spotted with cDNA in 50% DMSO are UV cross-linked at ~120 mJ. 4.1.2 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. 4.1.3 Place the printed slide(s) to be used for the hybridization in a Coplin jar containing preheated prehybridization buffer and incubate at 42oC for 45 minutes. 4.1.4 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. 4.1.5 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. 4.1.6 Use slides immediately following prehybridization to ensure optimal hybridization efficiency. 4.2 Hybridization 4.2.1 Prepare 1X hybridization buffer (50% formamide, 5X SSC, and 0.1% SDS). 4.2.2 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 mg/mL. 4.2.3 Prepare COT1-DNA (stock conc.1mg/mL) 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 20mg/mL. 4.2.4 Resuspend labeled probe (Cy3/Cy5 probe mixture: see SOP-M004) in 24 mL of 1X hybridization buffer. Note: Expose Cy labeled probe to light as little as possible during the hybridization process. 4.2.5 To block nonspecific hybridization add: COT1-DNA (20 mg/mL) ... 1mL Poly(A)-DNA (20mg/mL) ... 1mL Note: The COT1-DNA is organism specific: add mouse COT1 to labeled mouse probes and human COT1-DNA to labeled human probes. 4.2.6 To denature, heat the probe mixture at 95oC for 3 minutes and snap cool on ice for 30 sec. 4.2.7 Centrifuge the probe mixture at maximum angular velocity for 1 minute. Keep at room temperature and use immediately. 4.2.8 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 mL) 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 coverslips 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. 4.2.9 To the small wells at each end of the chamber add 10 mL of water (20mL total), cover, and seal the chamber. 4.2.10 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: 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. 4.2.11 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. 4.2.12 After the incubation remove foil and unseal hybridization chamber. Remove the slide from the chamber, taking care not to disturb the coverslip. 4.2.13 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. 4.2.14 After the coverslip is removed place slide in a staining dish containing lower stringency wash buffer (preheated to 42oC) and agitate for 4 minutes. 4.2.15 Wash the slide in a staining dish with high stringency wash buffer by agitating for 4 minutes at room temperature. 4.2.16 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.) 4.2.17 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. 4.2.18 Place slides in a light tight slide box until they can be scanned, preferably as soon as possible.Labeling - Title: Aminoallyl labeling of RNA for microarrays: SOP_M004_2. Description: THE INSTITUTE FOR GENOMIC RESEARCH Standard Operating Procedure TITLE: AMINOALLYL LABELING OF RNA FOR MICROARRAYS SOP #: M004 REVISION LEVEL: 2 EFFECTIVE DATE: 3/4/02 AUTHOR: Jeremy Hasseman PRIMARY REVIEWERS: Emily Chen, Ivana Yang 1. 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. 2. SCOPE This procedural format is utilized by Human Colon Cancer and Mouse microarray projects under the supervision of John Quackenbush within the Eukaryotic Genomics Dept. 3. MATERIALS 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/?L) (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) 3.9 Microcon YM-30 Centrifugal Filter Devices (Millipore; Cat # 42410) 4. REAGENT PREPARATION 4.1 Phosphate Buffers 4.1.1 Prepare 2 solutions: 1M K2HPO4 and 1M KH2PO4 4.1.2 To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine: 1M K2HPO4 ..9.5 mL 1M 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 100mM 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 -20oC. 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 4.3.1 Store unused solution at -20oC. 4.4 Sodium Carbonate Buffer (Na2CO3): 1M, 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 ?L 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 -80oC. 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. 5. PROCEDURE 5.1 Aminoallyl Labeling 5.1.1 To 10 ?g of total RNA (or 2 ?g poly(A+) RNA) which has been DNase I-treated and Qiagen RNeasy purified, add 2 ?L Random Hexamer primers (3mg/mL) and bring the final volume up to 18.5 ?L with RNase-free water. 5.1.2 Mix well and incubate at 70oC 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 ?L 0.1 M DTT .. 3 ?L 50X aminoallyl-dNTP mix .. 0.6 ?L SuperScript II RT (200U/?L) .. 2 ?L 5.1.5 Mix and incubate at 42oC for 3 hours to overnight. 5.1.6 To hydrolyze RNA, add: 1 M NaOH 10 ?L 0.5 M EDTA 10 ?L mix and incubate at 65oC for 15 minutes. 5.1.7 Add 10 ?L of 1 M HCl to neutralize pH. (Alternatively, one can add 25 ?L 1 M HEPES pH 7.0 or 25 ?L 1 M Tris pH 7.4) 5.2 Reaction Purification I: Removal of unincorporated aa-dUTP and free amines (use either the Qiagen or the Microcon 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 and 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 ?L (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 ?L 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 ?L 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 ?L. 5.2.10 Dry sample in a speed vac. or~ Microcon YM-30 Cleanup Method: 5.2.1 Place the Microcon sample reservoir into a collection microfuge tube. Add 375 ?L of water to the cDNA reaction tube and then pipette the sample into the Microcon sample reservoir/collection microfuge tube without touching the membrane. 5.2.2 Centrifuge at 12,000 rpm for 6-10 min. Note: Never centrifuge column to dryness as this will decrease product recovery. Adjust spin time to allow for optimal filtration while allowing enough solution to remain for sufficient recovery. 5.2.3 Empty collection microfuge tube. 5.2.4 To wash add 450 ?L of water to the sample reservoir/collection microfuge tube and centrifuge at 12,000 rpm for 6-10 minutes. 5.2.5 Empty collection microfuge tube and repeat previous wash step. 5.2.6 Invert Microcon sample reservoir into a new collection microfuge tube and centrifuge at 12,000 rpm for 1 minute to collect purified sample. 5.2.7 Dry the sample in a speed vac. 5.3 Coupling aa-cDNA to Cy Dye Ester. 5.3.1 Resuspend aminoallyl-labeled cDNA in 4.5 ?L 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 ?L 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 ?L 100 mM NaOAc pH 5.2. 5.4.2 Add 250 ?L (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 0.75 mL 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. 5.4.6 Place column in a clean 1.5 mL microfuge tube and carefully add 30 ?L 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 ?L. Note: This protocol is modified from the Qiagen QIAquick Spin Handbook (04/2000, pg. 18). 5.5 Analysis of Labeling Reaction 5.5.1 Use a 50 ?L Beckman quartz MicroCuvette to analyze the entire undiluted 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 (uL) * 37 ng/?L * 1000 pg/ng] 324.5 pg/pmol Note: 1 OD260 = 37 ng/?L 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 (?L) 0.15 pmol Cy5 = OD650 * volume (?L) 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) 5.5.8 After analysis mix together the two differentially labeled probes (Cy3 vs. Cy5) which will be hybridized to the same microarray slide. 5.5.9 Dry the Cy3/Cy5 probe mixture to completion in a speed vac and continue with SOP: M005 for the hybridization of the probe to a microarray slide.MIAME ScoreRaw DataOrganizationAssays and DataMAGE-TAB FilesArray DesignsYonghong WangJohn QuackenbushJoseph WhiteShuibang WangfalseTranscription profiling of asthma induced mice treated with LPS in a controlled environmentExamination of gene expression associated with asthma induced by treatment of mice with LPS in a controlled environment. Related experiment E-TIGR-82005-01-01T00:00:00Z2021-10-08T11:14:59Z2021-10-08T11:14:59ZE-TIGR-7EFO_0002768