{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Norman Lee"],"study_type":["transcription profiling by array"],"organism":["Rattus rattus"],"species":["Rattus rattus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-TIGR-79"],"description":["Examination of gene expression associated with hypoxia treatment of parental salt sensitive (SS) and consomic Brown Norway (SSYBN) rat strain. SSYBN rats are derived from introgression of chromosome Y from BN rats into the SS genetic background."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Title: MCW_RNA_preparation. Description:  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.","Labeling - Title: 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."],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","MAGE-TAB Files","Array Designs"],"pubmed_authors":["Anne Kwitek","Norman Lee","Hongying Wang","Joseph White"],"additional_accession":[]},"is_claimable":false,"name":"Transcription profiling of rat liver response to hypoxia treatment of parental salt sensitive (SS) and consomic Brown Norway (SSYBN) rat strain","description":"Examination of gene expression associated with hypoxia treatment of parental salt sensitive (SS) and consomic Brown Norway (SSYBN) rat strain. SSYBN rats are derived from introgression of chromosome Y from BN rats into the SS genetic background.","dates":{"release":"2005-01-01T00:00:00Z","modification":"2022-03-14T16:20:34.992Z","creation":"2022-03-14T16:20:34.992Z"},"accession":"E-TIGR-79","cross_references":{"EFO":["EFO_0002768"]}}