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The Laboratory of J. Michael Bishop at the University of California, San Francisco
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| This protocol can be used to quantitate an mRNA of interest if the RNA probe is present in molar excess over the target mRNA. A labeled RNA transcript is hybridized to a population of mRNAs and any non-hybridized (single stranded) RNAs are digested by RNase A and RNase T1. The hybridized mRNA is then analyzed on an urea/polyacrylamide gel. |
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A. Probe DNA Cloning and Isolation
1. Subclone probe fragment into an appropriate vector (such as pGem vectors). Amplify subcloned plasmid DNA (see DNA Miniprep Isolation Protocol)
2. Linearize the amplified plasmid with an appropriate Restriction Enzyme.
3. To the aqueous phase, add an equal volume of Phenol:Chloroform, mix well by inversion, microcentrifuge to separate phases and save the aqueous phase (upper layer).
4. To the aqueous phase, add an equal volume of 100% Ethanol and centrifuge in a microcentrifuge at full speed for 5 minutes to pellet the DNA. (see Hint #1)
5. Discard the supernatant, invert the tubes over a paper towel, and allow the pellet to air dry for a couple of minutes.
6. Redissolve the DNA pellet at approximately 100 ng/μl in 10 mM Tris, pH 8.0.
B. Transcription and Radiolabeling of RNA Probe
1. Using a Riboprobe kit (see Hint #2):
Add the following reagents in the order given and at room temperature
Approximately 100 ng of the Linearize DNA
5 μl of 5X Transcription Mix
2 μl of 0.1 M DTT
1 μl (20 to 40 Units) of RNasin
1 μl of 10 mM ATP
1 μl of 10 mM CTP
1 μl of 10 mM GTP
100 μCi of α-[32P]-UTP
1 μl T7 RNA polymerase
Add DEPC-treated distilled deionized water (DEPC-ddH2O) to 25 μl final volume.
2. Incubate for 30 minutes to 1 hour at 37°C.
3. Add Formamide loading dye solution to reaction.
4. Incubate at 85°C for 2 minutes.
5. Load sample onto a 7 M Urea/7% Acrylamide Gel and electrophoresis until dye buffer is near the bottom of the gel (see protocol on Electrophoresis of RNA through urea/acrylamide gels) (see Hint #3).
6. After disassembling gel apparatus, carefully transfer the Acrylamide gel to a container that has been rinsed with DEPC-treated water or baked overnight. Soak gel in 0.5 μg/ml Ethidium Bromide for 15 minutes. Soak gel in DEPC-treated water for 10 minutes to briefly destain the gel.
7. Cut out radiolabeled RNA band from the gel using a new razor blade.
8. Elute RNA from gel by incubating in Gel Elution Buffer for a few hours at room temperature (see Hint #4).
9. Centrifuge the solution briefly to pellet gel fragments and collect the supernatant.
10. To the supernatant, add an equal volume of Phenol:Chloroform, mix well by inversion, centrifuge in a microcentrifuge on full speed for 5 minutes to separate phases and save the aqueous phase (upper layer).
11. To the aqueous phase, add an equal volume of 100% Ethanol, mix by inversion, and incubate on dry ice for 10 min. Microcentrifuge the tube at top speed for 10 min to pellet the RNA.
12. Discard the supernatant and allow the pellet to air dry for a couple of minutes.
13. Redissolve the RNA pellet in Hybridization Mix.
C. Hybridization
1. To a total RNA sample (2 to 40 μg) add 100% Ethanol, mix by inversion, and incubate for 10 min on dry ice to precipitate the RNA. Microcentrifuge for 10 min at top speed to pellet the RNA (see Hint #1).
2. Dilute the radiolabeled RNA probe in Hybridization Mix such that 15 μl contains approximately 100 cpm (using a hand survey meter).
3. Add 15 μl of the radiolabeled probe to the pelleted RNA sample from Step (C1) and mix thoroughly to completely dissolve the RNA.
4. Incubate at 85°C for 2 minutes to denature any secondary structure in the RNA.
5. Immediately transfer the tubes to a water bath set to the desired hybridization temperature (see Hint #5).
6. Incubate from 12 to 24 hours.
D. RNase Digestion
1. Prepare RNase solution.
2. Add 300 μl of RNase solution to each hybridization sample. Add the RNase solution to the samples while they are still incubating at the hybridization temperature after completion of Step (C6).
3. Remove tube from water bath, vortex immediately, and transfer to an ice-cold water bath until all the samples have received the RNase solution.
4. Quickly remove all tubes from the ice bath and incubate the tubes at 30 to 37°C for 30 minutes.
5. Add 20 μl of 10% SDS and 5 μl of Proteinase K Solution.
6. Incubate the tubes at 37°C for 15 minutes.
7. To the reaction mixture, add an equal volume of Phenol:Chloroform, mix well by inversion, centrifuge in a microcentrifuge for 5 minutes on full speed to separate the phases and save the aqueous phase (upper layer).
8. To the aqueous phase, add an equal volume of 100% ethanol and centrifuge in a microcentrifuge for 5 minutes at full speed for 10 min to pellet the RNA.
9. Discard the supernatant and allow the pellet to air dry for a couple of minutes.
10. Redissolve the RNA pellet in Formamide loading dye.
11. Incubate at 85°C for 2 minutes.
12. Load RNA sample on 7 M Urea/5 to 10% Acrylamide Gel and electrophorese. After the gel has finished electrophoresis, dry down the gel. Expose the gel tom X-Ray film or on a phosphoimager screen to visualize. (see protocol on Electrophoresis of RNA through urea/acrylamide gel).
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| Gel Elution Buffer |
| 0.5% (w/v) SDS
1 mM EDTA
0.5 M Ammonium Acetate
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| 7 M Urea/7% Acrylamide Gel |
| Urea M.W. = 60.06
1X TBE
7% (w/v) Acrylamide (CAUTION See Hint #6)
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| Formamide Loading Dye |
| 80% (v/v) Formamide (CAUTION See Hint #6)
10 mM EDTA, pH 8.0
1 mg/ml Xylene Cyanol FF
1 mg/ml Bromphenol Blue
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| T7 RNA Polymerase |
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| RNasin |
| 20 to 40 Units/μl RNasin
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| 0.5 μg/ml Ethidium Bromide (CAUTION See Hint #6) |
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| Ethanol |
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| α-[32P]-UTP |
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| α-[32P]-UTP |
| 10 μCi/μ α-[32P]-UTP (1,400 Ci/mmol; CAUTION See Hint #6)
Need approximately 100 μCi
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| 10 mM GTP |
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| 10 mM CTP |
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| 10 mM ATP |
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| 0.1 M DTT |
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| 10 mM Tris, pH 8.0 |
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| Phenol:Choloform |
| 25:24:1 Phenol:Chloroform:Isoamyl Alcohol (CAUTION See Hint #6)
Store at 4°C in dark glass bottle.
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| 10 mg/ml Proteinase K |
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| 10% (w/v) SDS |
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| RNase Solution |
| 2 μg/ml RNase T1
Keep solution ice cold until used
Requires 300 μl per reaction tube
Prepare just before use in digestion buffer
40 μg/ml RNase A
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| Digestion Buffer |
| 5 mM EDTA
300 mM NaCl
10 mM Tris, pH 7.5
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| Hybridization Mix |
| 1 mM EDTA
80% (v/v) Formamide
400 mM NaCl
40 mM PIPES, pH 6.4
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Isoamyl Alcohol
Chloroform
EDTA
Restriction Enzyme
Phenol
Sodium Chloride
Iń-[32P]-UTP
Proteinase K
Ethidium Bromide
Riboprobe Kit
DEPC-treated ddH2O
Tris
Bromophenol Blue
GTP
CTP
RNasin
Formamide
RNase, T1
Urea
Ammonium Acetate
Ethanol
DTT
Acrylamide
Xylene Cyanol FF
ATP
RNase A
SDS
RNA Polymerase, T7
PIPES
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1. If necessary, adjust salt concentration to 200 mM NaCl or 300 mM Sodium Acetate.
2. To minimize degradation of the RNA by RNAses, wear gloves when handling samples and reagents, use sterile plasticware, and use DEPC-treated water and buffers.
3. The electrophoresis conditions will depend on the size of your RNA probe
4. Length of time may vary so determine empirically
5. Hybridization temperature must be determined empirically. A good test range is from 42 to 60°C. Most probes will hybridize at approximately 45°C.
6. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
OVERVIEW / GENERAL COMMENTS
This protocol can be used to quantitate an mRNA of interest if the probe is present in molar excess over the target mRNA. The probe is purified by Acrylamide Gel eletrophoresis in this protocol.
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