Microarray Protocol
PhysGen
Gretta Borchardt
with
Anne E. Kwitek, Ph.D.
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% salt 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® (plastic restraint cones)
Labeled 50ml tubes containing RNAlater
Labeled 1.5ml tubes for liver DNA sample
Dissecting tools
Saline
Paper towels
Liquid Nitrogen
Guillotine
2) Fasting begins at 9PM. Tissue collection begins at 9AM 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 tracking form (see example on 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, left kidney, one liver lobe, and two lung lobes into individually labeled tubes (each containing 12-15ml of RNAlater™,) with no pieces larger than 0.5cm in one direction. (A liver sample is also placed in a 1.5ml screw top tube for DNA archives at MCW.)
d) Remove the transponder from the rat and dispose of the carcass in the ARC burn cooler.
e) Rinse dissecting tools with sterile saline between tissues and 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 placed in a 4°C refrigerator following harvest. If RNA extraction does not take place within 1 month, tissues are stored with RNAlater™ at -20°C.
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) For liver and kidney samples, 500mg of tissue is removed the RNAlater and placed in a 50ml conical tube containing 5ml of Trizol reagent™ (1ml of Trizol per 100mg of tissue. For heart and lung samples, 1000mg of tissue and 10ml of Trizol is used. Extra tissue samples are removed from the RNAlater and archived at -80°C.
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, and then incubate at room temperature for 5 minutes.
4) Spin the samples at 10,000Xg, 4-8°C, for 20 minutes.
5) Transfer aqueous (top) layer to a new 15ml tube and add 0.5ml isopropanol per 1ml of Trizol. Incubate samples at room temperature for 10 minutes, and then spin for 30 minutes at 4000Xg at 4-8°C.
6) Discard the isopropanol. Wash the pellet (containing RNA) with 2.5ml of 75% ethanol by spinning at 4000Xg for 5 minutes, 4-8°C.
7) Discard the ethanol and air-dry the tubes upside down for 10 minutes to evaporate ethanol.
8) Re-suspend the pellet in 1.0ml Diethylpyrocarbonate (DEPC) treated water and place in labeled microcentrifuge tubes.
9) Warm to 55°C for 10 minutes.
10) Measure the samples spectrophotometrically at 260/280nm to obtain RNA concentration.
11) 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, columns and tubes, while the 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 / Final vol. of RNA sample and DEPC H20 / Buffer RLT / Ethanol<500mg / 500ml / 2.0ml / 1.4ml
500-1000mg / 1000ml / 4.0ml / 2.8ml
1) Calculate the volume of RNA sample required for 1000mg (maximum) of RNA to be cleaned and bring sample up to volume with DEPC water, as indicated in chart. (In cases where 1000mg is not possible, use the maximum attainable.)
2) Add Buffer RLT, according to chart above, and mix.
3) Add 100% ethanol and shake, and then apply 4ml of the sample to the RNeasy midi column in the accompanying 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. Add another 2.5 ml RPE, and repeat centrifuge for 5 min.
7) Place column in a new 15ml tube and add 250ml DEPC water, let stand 1 min and centrifuge for 3 minutes to elute RNA (use only 150ml water if sample is less than 150mg RNA.)
8) For increased yield, repeat the elution and centrifuge as in step 7. The first elute can be used for increased concentration, but correspondingly, the yield will be decreased.
9) The concentration of the samples is measured spectrophotometrically at 260nm/280nm.
10) To assess quality, 1mg of sample is run on a 1% agarose gel. (Gel images are stored at MCW.)
11) Clean RNA is stored at -80°C in labeled 2ml tubes until shipment.
12) Samples are 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)
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