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.
Phenotyping Protocol for Mouse tissue, strain and sex variation BrainSexStrain, LungSexStrain posted to date Title: Collecting Tissues to Test for Inter-sex and Inter-strain Variation in Gene Expression Experiment Number: GX 01-02 Protocol Prepared: 01/08/2001BP; revised 02/21/2001 by KS (JAX), and by BP on 10/17/02 (BP is Bev Paigen and KS is Karen Svenson of The Jackson Laboratory) 0.0 Question: What genes are expressed in each of the nine tissues? 1.0 Background: Gene expression in mouse tissues may vary between sexes and among mouse strains. We will assess this variation in nine different mouse tissues from the males and females of three different mouse strains. 2.0 Phenotypes to be investigated: none 3.0 Tissues to be collected: Heart, lung, liver, kidney, brain stem, brain cortex, intestine, aorta, and reticulocytes 4.0 Overall experimental design: Harvest tissues from 10 males and 10 females from 3 inbred strains; C57BL/6, A/J, and DBA/2; that are 10 ± 1 weeks old. Compare the strain and sex-specific gene expression in each tissue. 5.0 Detailed experimental design: To decrease inter-mouse variation, pool RNA samples. Until Experiment GX01-01 (designed to determine how many mice must be pooled to decrease inter-mouse variation to the same level as inter-chip variation) is complete, pool tissues from groups of 10 mice (Jackson Laboratory statistician Dr. Churchill estimates that tissues from six mice need to be pooled; as a precautionary measure, pool 10). To harvest tissues as soon as possible after the mice die, harvest them in groups from lots of 10 mice of the same sex and strain as in the following example: Group A: Flush (see Notes, sec. 11.0) ten male C57BL/6 mice and harvest their lung, heart, liver, and kidney tissues in that order. Group B: Decapitate ten other C57BL/6 males and harvest their brain stem and cortex tissues separately. Group C: Flush intestine, and collect it and the aorta (from heart to iliac bifurcation) of. Group D: Treat 10 other C57BL/6 males with phenyl hydrazine (SIGMA cat. # P6926). Give each mouse 0.3ml [0.25% stock; 0.125g/50ml ddH20] each day for five days. Rest each mouse for two days before sampling its reticulocytes. Repeat the tissue sampling procedure outlined above for lots of ten C57BL/6 females, 10 A/J males, and so on. Before harvesting their tissues, fast mice for four hours, from 6 to 10 am. Harvest tissues from 10 to 11 am, slice them into pieces no larger than 0.5cm in any direction, and place them into 15 ml Falcon tubes containing RNA later (Ambion, Inc.). (See RNA Extraction Protocol/Version 1.0 for details) To insure that the gene expression pattern of each tissue can be adequately determined with gene arrays, send at least five times 80 µgms (preferably 120) of RNA for each tissue. Store either the remainder of the RNA at The Jackson Laboratory. Collect all tissues by the end of February, 2001, and send RNA samples to Dr. Quackenbush by March 12, 2001. 6.0 Animal husbandry 6.1 Feed: Laboratory chow (Purina 5K52 - NIH31, Purina, St-Louis, MO) 6.2 Water: Water from Bar Harbor Water Co. is filtered through bag filters and acidified to pH of 2.8 to 3.1 by using HCL. Sodium menadione bisulfite (Vitamin K analog) is also added to the water at a concentration of 0.43 to 0.47 ug/ml. 6.3 Bedding: Pine shavings (Washington Junction, Ellsworth, ME 04605) 6.4 Photoperiod: 12-h light/12-h dark cycle (7am-7pm) 6.5 Barrier Facility? Yes 6.6 Pathogen Free? Yes (no Helicobacter, Pneumocystis, Pasturella, which can be found at The Jackson Laboratory. For a complete list of pathogens tested for and those absent from The Jackson Laboratory,see the Jax web page (www.jax.org). 6.7 Caging/ventilation: individually ventilated cages 7.0 Timing:. Collect all tissues by the end of February, 2001, and send RNA samples to Dr. Quackenbush by March 12, 2001. (Shipped Feb 21, 2001) 8.0 RNA details: See RNA Extraction Protocol/Version 1.0 for details. Isolate RNA from each animal separately. Pooling will be done at TIGR. 9.0 Array design and Array analysis strategy: 10.0 Data to be collected for analysis: not applicable 11.0 Notes: Mice should be 10 ± 1 weeks old before their tissues are harvested. Flush mice before their lungs, livers, hearts, and kidneys are harvested. Insert the needle of a 60cc syringe containing 0.9% saline through the left ventricle of the heart and flush using constant pressure. This procedure takes less than one minute per mouse.
Follow manufacturers instructions.
THE INSTITUTE FOR GENOMIC RESEARCH Standard Operating Procedure TITLE: MAKING MICROARRAY PRINTING PLATES (IN DMSO) SOP #: M003 REVISION LEVEL: 1 EFFECTIVE DATE: AUTHOR: Jeremy Hasseman PRIMARY REVIEWERS: Emily Chen, John Quackenbush, Ivana Yang 1. PURPOSE This protocol describes the method for making microarray printing plates in a 96 well format. (The same procedure applies to a 384 well format as well.) 2. MATERIALS 2.1 Costar 96 well V-bottom plates (Corning; Cat # 3897) 2.2 Costar Corner Notch Lid (Corning; Cat # 3930) 2.3 Dimethyl Sulfoxide (DMSO) 3. PROCEDURE 3.1 Pipette 5 ?L of DMSO into the bottom of a Costar V-bottom plate. 3.2 Label the plates according to the orientation the plates will have in the array printer. Note: TIGR Intelligent Automation Systems (IAS) arrayer hotels hold plates with the A1 short-side facing outward, therefore, that side is the most convenient to have labeled. 3.3 Pipette 5 ?L of purified PCR product into the DMSO and mix with pipette. 3.4 Centrifuge at 2700 rpm for 1 minute to make sure the 50% DMSO/ PCR product solution is in the bottom/center of the well where the spotting pins will dip into the well. 3.5 Store in 4o C for a short term storage and -20o C for long term storage. Note: DMSO has an extremely low evaporation rate therefore sealing the plates with tape is NOT necessary. Residual adhesive may complicate lid removal during an array printing.