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MODIFIED EBERWINE ("ANTISENSE") RNA AMPLIFICATION PROTOCOL FOR MICROARRAY ANALYSIS OF GENE EXPRESSION
Modified Eberwine ("Antisense") RNA Amplification Protocol for Microarray Analysis of Gene Expression
Overview
DNA microarrays comprise an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slides or other solid substrates. The expression of genes in cells can be monitored with microarrays by hybridization of fluorescently-labeled RNA probes to the DNA molecules in the array. This protocol describes a method to amplify cellular RNA for use in microarray experiments. Total RNA or messenger RNA (mRNA) is first subjected to reverse transcription to create complementary DNA (cDNA). Amplification of RNA is accomplished through in vitro transcription of the cDNA. The amplified RNA can then be used to synthesize fluorescent probes.
Procedure
A. First Strand Synthesis (see Hint #1)
1. Mix 20 to 100 nanograms of mRNA isolated from cells to be analyzed, Trehalose, and DEPC-ddH2O in a final volume of 9 μl and add 1 μl of "Eberwine" Oligo-dT/T7 Primer (see Hint #2).
2. Incubate the mixture at 65°C for 10 min and then place the tube on ice.
3. Add the following components to the tube:
4 μl of 5X First Strand Buffer
2 μl of 0.1 M DTT
1 μl of RNAsin
1 μl of dNTP Mix
1 μl of Linear Acrylamide
1 μl of Superscript II
4. Perform polymerase chain reaction (PCR) in a thermocycling machine under the following reaction conditions:
1 cycle of 37°C for 5 min
1 cycle of 45°C for 5 min
10 cycles of:
60°C for 2 min
55°C for 2 min
5. After completion of the PCR reaction, place the tube on ice and keep it cool while adding second strand components.
B. Second Strand Synthesis (see Hint #1)
1. Add the following components to the tube from Step #A5:
106 μl of DEPC-ddH2O
15 μl of 10X Second Strand Buffer
3 μl of dNTP Mix
1 μl of E. coli DNA Ligase
4 μl of E. coli DNA Polymerase I Holoenzyme
1 μl of RNase H
2. Incubate the reaction at 16°C for 2 hr.
3. If the amplification was performed with mRNA, stop the reaction by adding 10 μl of 0.5 M EDTA and incubate the reaction at 65°C for 10 min.
4. If the amplification was performed with total RNA, stop the reaction by adding 7.5 μl of 1 M NaOH/2mM EDTA and incubate the reaction at 65°C for 10 minutes (see Hint #3).
C. Sample Extraction and Precipitation
1. Add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) to the reaction.
2. Mix by vortexing and then centrifuge at maximum speed for 5 min in a microcentrifuge to separate the phases.
3. Transfer the upper (aqueous) phase to a fresh 1.5 ml microcentrifuge tube (see Hint #4).
4. Add 70 μl of 7.5 M Ammonium Acetate and 1 ml of -20°C 100% Ethanol.
5. Mix by vortexing and then centrifuge immediately for 20 min at maximum speed in a microcentrifuge at room temperature (see Hint #5).
6. Wash with 100 μl of 100% Ethanol and centrifuge at maximum speed in a microcentrifuge for 5 min (see Hint #6).
7. Remove all excess Ethanol and dry the pellet briefly at room temperature (see Hint #7).
8. Resuspend the pellet in 10 μl of ddH2O. At this point, the cDNA may be stored for long periods of time at -20°C or -80°C.
9. Prepare Sephadex G75 spin columns (Pharmacia) by resuspending 3 g of Sephadex G75 powder in 50 ml of DEPC-TE. Pack a 1 cc syringe---that has been plugged with glass wool---with Sephadex G75 by performing the following steps:
Fill the syringe with Sephadex G75 and place the syringe in a microcentrifuge
Place the syringe/microcentrifuge combination into a 15 ml conical tube
Centrifuge at 700 X g for 2 min to remove excess buffer from the column
Discard the flow-through and add Sephadex G75 again
Repeat this procedure until the column volume of Sephadex G75 is between 0.8 ml to 1 ml.
10. Place the column in a fresh microcentrifuge tube and add the resuspended cDNA to the Sephadex G75 column (see Hint #8).
11. Centrifuge at 700 X g for 5 min to collect the flow-through.
12. Lyophilize the collected cDNA until the sample reaches a volume of 16 μl or less.
D. In Vitro Transcription
1. Transcribe RNA from the cDNA with an Ambion T7 Megascript Kit (Ambion) or equivalent. Double the reaction volume (40 μl) and perform the reaction with the Megascript Kit at 37°C for 4 hr.
2. Extract the transcribed RNA with Phenol:Chloroform:Isoamyl Alcohol as described in Steps #C1 through #C3.
3. Apply the extracted RNA to a ChromaSpin TE+30 column (Clontech) and centrifuge at 700 X g for 5 min.
4. Transfer the flow-through to an RNase-free, 1.5 ml microcentrifuge tube.
5. Lyophilize the sample until the volume is 13 μl or less.
6. Adjust the volume to 13 μl with ddH2O if necessary.
7. Quantitate the approximate yield by electrophoresis of 1μl of the sample through a 1% (w/v) Agarose gel (see Protocol ID# 1265) and/or by spectrophotometry at 260nm wavelength of a 1:50 or 1:100 dilution of the sample (see Protocol ID #9023; see Hint #9).
8. Proceed with the synthesis of fluorescent probes from the RNA (see Protocol ID #2254 or Protocol ID #2255).
Solutions
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Add 150 μl of 100% Ethanol
0.1 μl of 0.5 M EDTA
0.5 μl of 3 M Sodium Acetate
1.5 μl of 2 M Tris-Cl, pH 8
Dilute 1:100 in DEPC-ddH2O prior to use
2 M Tris-Cl, pH 8
Centrifuge 5 min at top speed in a microcentrifuge
73 μl of ddH2O
Polymerize for 30 min
1 μl of 10% (w/v) Ammonium Persulfate
Aspirate the supernatant and dissolve in 375 μl of DEPC-ddH2O
3.75 mg Acrylamide
0.1 μl of TEMED
CAUTION! See Hint #10
DEPC-TE
Treat with DEPC as described in the BioTools section of this website
10 mM Tris-Cl, pH 8.0
1 mM EDTA
7.5 M Ammonium Acetate
Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
CAUTION! See Hint #10
1 M NaOH/2 mM EDTA
1 M Sodium Hydroxide (CAUTION! See Hint #10)
2 mM EDTA
Second Strand Buffer (10X)
900 mM KCl
1.5 mM Nicotine Adenine Dinucleotide (Calbiochem)
46 mM Magnesium Chloride
100 mM (NH4)2SO4
200 mM Tris-Cl, pH 6.9
0.5 M EDTA
3 M Sodium Acetate
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
Linear Acrylamide (10 μg/μl)
"Eberwine" Oligo-dT/T7 Primer
1 mg/ml Oligonucleotide: 5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC TTT TTT TTT TTT TTT-3'
RNase H (Gibco/BRL)
2 Units/μl RNase H
E. coli DNA Polymerase I--Holoenzyme (NEB)
10 Units/μl E. coli DNA Polymerase I--Holoenzyme
E. coli DNA Ligase (NEB)
10 Units/μl E. coli DNA Ligase
Superscript II (Gibco/BRL)
Superscript II Reverse Transcriptase
dNTP Mix (Pharmacia)
10 mM each dATP, dCTP, dTTP, dGTP
RNasin (Gibco/BRL)
DTT (Gibco/BRL)
Supplied with Superscript II Enzyme
0.1 M DTT
First Strand Buffer (5X) (Gibco/BRL)
Supplied with Superscript II Enzyme
Trehalose (Sigma)
Prepare in ddH2O
1.7 M Trehalose
BioReagents and Chemicals
Acrylamide
Sodium Hydroxide
E. coli DNA Polymerase I Holoenzyme
RNase H
Tris
EDTA
Isoamyl Alcohol
Chloroform
Phenol
Agarose
Superscript II
Ammonium Sulfate
RNasin
Hexamers
Nicotine Adenine Dinucleotide
Trehalose
Ethanol
dNTP mix
E. coli DNA Ligase
Ammonium Acetate
DTT
Sodium Acetate
Oligonucleotide
TEMED
Ammonium Persulfate
Protocol Hints
1. The contributor of this protocol suggests that the first and second strand synthesis reactions should be performed in RNase-free PCR tubes. Ensure that solutions and all manipulations of RNA are maintained RNase-free (see the BioTools section of this website for instructions). If total RNA is used instead of mRNA, add 1 to 3 μg of total RNA to the reaction.
2. RNA may be dried down in Speedvac to concentrate. Trehalose is viscous; mix well by pipetting.
3. The addition of this solution results in the degradation of ribosomal RNA and tRNA, which can interfere with subsequent reactions.
4. The contributor of this protocol recommends the use of the Phase Lock reagent (5'-3' Inc.) during the organic extraction.
5. It is important to centrifuge immediately so as not to precipitate residual proteins or low molecular weight nucleotides.
6. At this point, a large pellet should be visible. If no pellet is visible, it is likely that the reagents were contaminated with RNase or the initial RNA was degraded.
7. If the pellet is too dry, it will be difficult to resuspend.
8. When loading the RNA onto the column, carefully add the sample to the center of the top of the column bed.
9. Optical density is less accurate than Agarose gel electrophoresis, and the quality of the transcribed RNA can be visualized on the gel.
10. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
Citation and/or Web Resources
4. Eberwine, et al., (1992) PNAS 89 :3010.
3. Carninci, et al., (1998) PNAS 95 :520.
5. Van Gelder, et al., (1990) PNAS 87 :1663.
1. Lab website
2. Luo, et al., (1999) Nature Medicine 5(1): 117.
