Protocols: Microarray


The Combination of TRIzol and QIAGEN
The Combination of TRIzol and phenol/chloroform
RNA Isolation for bone tissues
Solutions required for RNA Isolation Protocol
Tissue collection

 

The Combination of TRIzol and QIAGEN Procedure

** When working with TRIzol Reagent, work in fume hood and use gloves and eye protection. Use RNAse-Away plus water rinse on all glassware, tubes, forceps, etc.

A. Homogenization

1. Homogenize tissue samples in ~1 ml of TRIzol reagent per 50-100 mg of tissue using mortar and pestle with liquid nitrogen (or freezer mill). Sample volume should not exceed 10% of the TRIzol Reagent volume used for homogenization.
2. After homogenization, remove insoluble material from the homogenate by centrifuging at 10,000 RPM for 10 min at 4°C.
a. Transfer the top layer solution into a new tube;
b. Discard pellet.

B. Phase Separation

1. Incubate homogenized samples for 5 min at room temp. to permit complete dissociation of nucleoprotein complexes.
2. Add 0.2 ml of chloroform per 1 ml of TRIzol used for homogenization.
3. Cap sample tubes and shake vigorously by hand for 15 sec.
4. Incubate samples at room temp for 2-3 min. until sample starts separating into upper and lower phases.
5. Centrifuge samples at 10,000 RPM for 15 min at 4°C.
6. The RNA is in the top aqueous phase. Transfer top phase into new tube.

C. RNA Precipitation

1. Precipitate the RNA from the aqueous phase by mixing with isopropanol. Use 0.8 ml of isopropanol per 1 ml of TRIzol Reagent used for the original homogenization.
2. Incubate samples at room temp. for 10 min.
3. Centrifuge at 10,000 RPM for 10 min at 4°C.
4. There should be a pellet visible at the bottom of the tube.

D. RNA Wash

1. Remove and dispose of supernatant.
2. Wash RNA pellet once with 75% ethanol.

E. Redissolving the RNA pellet

1. Briefly dry the RNA pellet. Give a quick spin in microcentrifuge to collect all the liquid at the bottom of the tube for removal.
2. Dissolve RNA in Rnase-free water and then cleanup RNA using RNeasy spin column (QIAGEN Cat# 75142) as described here.

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The Combination of TRIzol and phenol/chloroform Procedure

** When working with TRIzol Reagent, work in fume hood and use gloves and eye protection. Use RNAse-Away plus water rinse on all glassware, tubes, forceps, etc.

A. Homogenization

1. Homogenize tissue samples using mortar and pestle with liquid nitrogen (or freezer mill) and then add ~5 ml of TRIzol reagent per 50-100 mg of tissue (Sample volume should not exceed 10% of the TRIzol Reagent volume used for homogenization).
2. After homogenization, remove insoluble material from the homogenate by centrifuging at 10,000 RPM for 10 min at 4°C.
a. Transfer the top layer solution into a new tube;
b. Discard pellet.

B. Phase Separation

1. Incubate homogenized samples for 5 min at room temp. to permit complete dissociation of nucleoprotein complexes.
2. Add 0.2 ml of chloroform per 1 ml of TRIzol used for homogenization.
3. Cap sample tubes and shake vigorously by hand for 15 sec.
4. Incubate samples at room temp for 2-3 min. until sample starts separating into upper and lower phases.
5. Centrifuge samples at 10,000 RPM for 15 min at 4°C 6. The RNA is in the top aqueous phase. Transfer top phase into new tube.

C. Phenol/chloroform extraction

1. add equal volume of Phenol/chloroform/IAA to the aqueous phase.
2. Shake for 10 sec, return to ice, and repeat this process for several time.
3. Centrifuge at 10,000 RPM for 10 min at 4°C.
4. Transfer top phase into a new tube.
5. Add equal volume of chloroform/AA to the aqueous phase and shake for 10 seconds (repeat).
6. Centrifuge at 10,000 RPM for 10 min at 4°C.
7. Transfer the top phase into a new tube.

D. RNA precipitation

1. Add 0.1X sample volume of 3M sodium acetate (pH 4.8). Then, add 2.5X volume of (sample + sodium acetate volume) of cold 95% ETOH (-20°). Mix thoroughly.
2. Place samples in -80° freezer for 1 hr.
3. Centrifuge for 20 min at 10,000 RPM at 4°C.

E. RNA Wash

1. Remove and dispose of supernatant.
2. Wash RNA pellet once with 75% ethanol.

F. Redissolving the RNA pellet

1. Briefly dry the RNA pellet. Give a quick spin in microcentrifuge to discard all the liquid at the bottom of the tube.
2. Dissolve RNA in Rnase-free water.

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RNA Isolation for Bone Tissues

Day 1: Organic Extraction:

1. Homogenize tissue with pestle in mortar containing liquid nitrogen add 3.5 ml guanidine RNA extraction buffer (add 7.5 ul ß-ME per ml of guanidine buffer just before use).
2. Place samples in microcentrifuge (MCF) tubes and briefly vortex.
3. Incubate samples in 37°C water bath for 30 min. Vortex every 10 min.
4. On ice, add 125 ul of 3M sodium acetate (pH 4.8) per ml of guanidine. Mix Briefly.
5. Add 1X sample volume of 5:1 phenol/chloroform w/ isoamyl alcohol to achieve phase separation (phenol has 2 layers, use bottom layer).
6. Mix and vortex for 10 min, keeping samples on ice between mixings. Centrifuge for 20 min at 10,000 RPM at 4°C.
7. Transfer top layer (aqueous layer) into new MCF tube. Discard bottom layer.
8. Repeat steps 5-7.
9. Add 1X sample volume of 24:1 chloroform/isoamyl alcohol (NO phenol). Mix thoroughly.
10. Centrifuge for 20 min at 10,000 RPM at 4°C.
11. Transfer top layer into new MCF tube ON ICE.
12. Add 2.5X sample volume of cold 95% ETOH (-20° freezer). Mix thoroughly.
13. Leave at -20° overnight.

DAY 2: Alcohol Washes
*** ALL PROCEDURES ARE DONE ON ICE!!!

1. Centrifuge for 20 min at 10,000 RPM at 4°C.
2. Place tubes on ice. Using a sterile pipette, aspirate off ETOH. Should be able to see RNA pellet.
3. Add 3-4 ml of cold 70% ETOH (-20°) to the pellet. Mix briefly.
4. Centrifuge for 5 min at 10,000 RPM at 4°C.
5. Aspirate off as much ETOH as possible. (Use pasteur pipette, then 0.5 ul pipette).
6. Dissolve pellet in DEPC H20 (amt. added depends on sample size, use as little DEPC as possible. For liver, at least 100 ul). Let pellet sit until it dissolves. If pellet remains after 30 min then use pipette to gently scrape it from wall of tube. After pellet dissolves, transfer solution to a clean eppendorph tube.
7. Add 0.1X sample volume of 3M sodium acetate (pH 4.8). Then, add 2.5X volume of (sample + sodium acetate volume) of cold 95% ETOH (-20°). Mix thoroughly.
8. Place samples in -80° freezer for 1 hr.
9. Centrifuge for 20 min at 10,000 RPM at 4°C.
10. Place samples on ice. Aspirate off ETOH.
11. Add 1 ml cold 70% ETOH (-20°). Mix briefly.
12. Centrifuge for 5 min at 10,000 RPM at 4°C.
13. Aspirate off as much ETOH as possible. Let samples sit uncovered in hood ~ 20 min to allow rest of ETOH to evaporate. Give tube a brief spin in centrifuge and aspirate off any remaining alcohol with 0.5 ul pipette.
14. Dissolve pellet in DEPC H20. (For liver, may need as much as 130 ul. Normally 80-100 ul for bone).
15. Measure RNA concentration. 1 OD260 = 40 ug.
16. Remove 5-10ug of RNA for gel analysis to determine quality of RNA.

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Solutions required for RNA Isolation Protocol

Grinding

A. 0.75 M Sodium Citrate (100 ml) [Fisher Catalog # S279-500]
1. Weigh 22.06 g NaCitrate.
2. Add 75 ml DEPC H2O.
3. Mix with stir bar.
4. PH to 7.0 with HCl.
5. Add DEPC H2O to bring to 100 ml.
6. Autoclave 20 min. (OR filter sterilize if in hurry).

B. 10% Sarcosyl (~30 ml)
1. Weigh 3 g N-lauroylsarcosine [Fisher Catalog # BP234-500].
2. Add 30 ml DEPC H2O.
3. Mix with stir bar.
4. Store in sterile container.

C. Guanidine RNA extraction Buffer (~60 ml)
1. In beaker, weigh 25 g of guanidine isothiocyanate (CH5N3CHNS) [Fisher Cataolog # BP221-250].
2. Add 29.3 ml DEPC H2O.
3. Add 1.76 ml 0.75 M NaCitrate (pH 7.0).
4. Add 2.64 ml 10% Sarcosyl.
5. Mix with stir bar.
6. Filter sterilize into sterile bottle. Wrap in foil. Store at room temp < 3 months.

Organic extraction

A. Chloroform/Isoamyl
1. Ratio of 24 parts chloroform to 1 part isoamyl (ex:4800 ul chloroform, 200 ul isoamyl).
2. Mix and store under hood.

B. Phenol/Chloroform/Isoamyl [Fisher Catalog #BP1754-400]
This is a 5:1 phenol:chloroform solution with Isoamyl alcohol. Use this solution for step 5 of the Organic Extraction Protocol.

C. 3M Sodium Acetate (pH 4.8) (100 ml) [Fisher Catalog # BP334-500]
1. Weigh out 40.82 g of Sodium acetate.
2. Add 60-70 ml DEPC H2O.
3. PH to 4.8 with acetic acid (IT TAKES A LOT, UP TO 20 ml!!!).
4. Add water to make 100 ml.

Notes
1. Total RNA in the amount of 5 -10ug should be provided at a minimal concentration of 1ug/ul
2. RNA OD260/280 ratio should be equal or greater than 1.8
3. All RNA provided to the Facility must be resuspended in RNase/DNase free dH20.

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Tissue collection procedures

Incorrect handling of tissue or cells prior to RNA isolation has two implications: degradation of RNA and altered gene expression profile. Therefore, the proper collection and handling of tissues are the first and often the most critical step in obtaining high quality, intact RNA and maintaining the in vivo levels of gene expression for microarray hybridization.

The guidelines for tissue collection:

  1. collect tissues as quickly as possible and always on ice;
  2. use freshly dissected tissues for RNA extraction, otherwise immediately store in liquid nitrogen and do not thaw out before use. For some tissues like bone, RNA will be degraded if the tissues are thawed out before RNA extraction;
  3. for dissecting procedures that require a longer period of time, dissection can be performed in RNAlater solution (AMBION, Cat# 7020. http://www.ambion.com/catalog/CatNum.php?7020 ). RNAlater is an RNA stabilization solution, which can effectively inactivate RNases and stabilize RNA with tissues. Collection of such samples in RNAlater solution significantly improves the integrity of total RNA.

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