Microarray Protocol
Gretta Borchardt
with
Anne E. Kwitek, Ph.D.
PhysGen Microarray Protocol-RNA sample Preparation Procedure. In a collaborative effort between the PhysGen (MCW) and TREX (TIGR) Programs for Genomic Applications, we are isolating tissues from the rat consomic strains to be used in microarray studies. The physiological and gene expression data generated by these PGAs can be combined to help facilitate gene discovery and better understanding of the pathobiology leading to heart, lung, and blood disorders.
I. Experimental design
· 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
II. Experimental protocol
· 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, 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.
A. Tissue Collection
1) Tissue collection is performed in the necropsy room of MCW’s Animal Resource Center (ARC). The following equipment is used in tissue collection setup:
Transponder reader
Decapicones®
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 (see page 6)
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™, 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.
B. RNA extraction
· Equipment used in RNA extraction:
Trizol™ 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™ 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.
C. RNA clean up
· The extracted RNA pool is run through Qiagen’s RNeasy midi kit to remove any remaining genomic DNA or degraded RNA. Kit contains buffers and tubes, user must supply 96-100% Ethanol, b-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:
Total RNA in sample / Add DEPC H20 to / Buffer RLT / Ethanol<500mg / 500ml / 2.0ml / 1.4ml
500-1000mg / 1000ml / 4.0ml / 2.8ml
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-
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 250ml DEPC water, let stand 1 min, centrifuge (as in s 3 min to elute RNA (use only 150ml water if sample is less than 150mg 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.
III. Supply Information
Ambion (phone # 800-888-8804)
RNAlater (catalog # 7021)
Biomedic Data Systems (302-628-4100)
Transponders (IMI-1000)
Transponder reader (DAS-5006)
Braintree Scientific (781-843-2202)
Decapicones (DC-200)
Invitrogen (760-603-7200)
Trizol (15596018)
Stoelting (630-860-9700)
Small animal decapitator (51330)
Qiagen (800-426-8157)
RNeasy midi kits (75144)
DNase (79254)
Microarray
Tissue Harvest Pool Tracking form
Protocol______
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Microarray Protocol
PhysGen
SSBN___ Hypoxic Date______
Comments
Pool 5a Male ______
______
______
Pool 5b Male ______
______
______
(Extra Male ______)
Pool 6a Female ______
______
______
Pool 6b Female ______
______
______
(Extra Female ______)
SSBN___ Normoxic Date______
Comments
Pool 1a Male ______
______
______
Pool 1b Male ______
______
______
(Extra Male ______)
Pool 2a Female ______
______
______
Pool 2b Female ______
______
______
(Extra Female ______)
SS Hypoxic Date______
Comments
Pool 7a Male ______
______
______
Pool 7b Male ______
______
______
(Extra Male ______)
Pool 8a Female ______
______
______
Pool 8b Female ______
______
______
(Extra Female ______)
SS Normoxic Date______
Comments
Pool 3a Male ______
______
______
Pool 3b Male ______
______
______
(Extra Male ______)
Pool 4a Female ______
______
______
Pool 4b Female ______
______
______
(Extra Female ______)
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