Modified Eberwine (antisense) RNA Amplification Protocol

Modified Eberwine ("antisense") RNA Amplification Protocol
Chris Barry, MD, PhD, Pat Brown Lab, 4/22/99
barry_c@cmgm.stanford.edu

The optimal range for amplifying from mRNA is 20-100 nanograms. The optimal
range for total RNA is 1-3 micrograms.

First Strand Synthesis:

(First and second strand synthesis reactions are performed in 0.2 mL RNase
free PCR tubes.)

mRNA, trehalose (Sigma #T-5251, make 1.7M stock in DEPC water)/DEPC water
to 9 uL (final concentration of 0.6M trehalose/20 uL) and "Eberwine"
oligo-dT/T7 primer (5'AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG
CGC T15-3')1 uL (1 ug/uL). May dry down RNA in Speedvac to concentrate.
Trehalose is viscous; mix well by pipeting.

Heat 65C for 10 minutes then put on ice.

Add 4 uL 5X First Strand Buffer (GibcoBRL, comes with Superscript II enzyme)
2 uL 0.1M DTT (GibcoBRL, comes with Superscript II enzyme)
1 uL RNAsin (GibcoBRL #15518-012)
1 uL 10 mM dNTP mix (Pharmacia #27-2035-01, resuspend in DEPC water)
1 uL linear acrylamide (0.1 ug/uL, Ambion #9520 or homemade--see recipe below)
1 uL Superscript II (reverse transcriptase, GibcoBRL #18064-014)

Thermocycle: 37C 5 min, 45C 5 min, then 10 cycles alternating between 60C 2
min and 55C 2min.

Place on ice and keep cool while adding second strand components.



Second Strand Synthesis:

Add to 1st strand reaction:
106 uL DEPC water
15 uL 10X Second Strand Buffer (recipe below)
3 uL 10 mM dNTP mix (dilute in DEPC water, Pharmacia #27-2035-01)
1 uL E. coli DNA Ligase (10 U/uL, NEB #205L)
4 uL E. coli DNA Polymerase I--holoenzyme (10 U/uL, NEB #209L)
1 uL RNase H (2 U/uL, GibcoBRL #18021-071)

Incubate 16C for 2 hours. If amplifying from mRNA, stop reaction with 10 uL
0.5M EDTA and incubating at 65C for 10 minutes. If amplifying from total
RNA, stop with 7.5 uL 1M NaOH/2mM EDTA and incubating at 65C for 10 minutes
(latter degrades ribosomal and tRNA that can interfere with subsequent in
vitro transcription reaction).



Sample Extraction and Precipitation:

Phenol:chloroform:isoamyl (25:24:1) extract once (use 0.5 ml "phase lock
gel" tubes from 5'-3' Inc, #p1-257178). Add organics (150 uL) directly to
PCR tubes, being careful not to spill. Mix by pipeting 5-10X, then transfer
slurry to Phase lock gel tubes. Spin 5 minutes maximum speed (15k x g) at
room temperature. Transfer aqueous phase to RNase free 1.5 ml eppendorf
tube.

Add 70 uL 7.5M ammonium acetate (in DEPC water, 0.2 micron filtered) to
aqueous phase, then 1 mL absolute ethanol (-20C).
Vortex, centrifuge immediately for 20 minutes, maximum speed, at room
temperature. It is important to spin right away so as not to precipitate
residual proteins or low molecular weight nucleotides (i.e., free nucs or
primer).
Wash with 100 uL absolute ethanol once, spin 5 minutes. At this point, a
large (salt) pellet should be visible; if no pellet is seen at all, suspect
RNase contamintion of reagents or degraded starting RNA.
Remove all excess ethanol, dry briefly at room temperature (not completely
or resuspension will be difficult).
Resuspend in 10 uL DEPC water. (May stop here indefinitely at -20C.)

Prepack Sephadex G75 spin columns (Pharmacia #17-0050-01, make slurry by
adding 3g of powder to 50 mL DEPC TE, use 1 cc syringes and glass wool
plug, prespin twice for 5 minutes, 700xG, room temperature, adding resin
between first and second spin for final packed resin volume=0.8-1.0 mL),
then pass cDNA (10 uL) over column for 5 minutes at 700xG. When loading
column, be careful to put the sample in the center of the matirx in order
to prevent wicking of sample onto side.
Lyophilize flow through (20-50 uL) to 16 uL or less in Speedvac.



In Vitro Transcription:

Ambion T7 Megascript Kit (#1334), double the standard 20 uL reaction (total
volume=40 uL), 37C for 4 hours. (Follow manufacturer's instructions
verbatim.)
Phenol:chloroform:isoamyl extract once (in 5'-3' phase lock gel tubes).
Pass over ChromaSpin TE+30 column (Clontech #K1321-2). Prepare columns
while performing the organic extraction: remove top cap then snap off
bottom; prespin 5 minutes at 700 x g, room temperature. When loading
column, be careful to put the sample in the center of the matirx in order
to prevent wicking of sample onto side.
Transfer flow through to Rnase free 1.5 mL Eppendorf tube.
Lyophilize to 13 uL or less.
Adjust volume to 13 uL DEPC water, if necessary.
Quantitate approximate yield by 1% agarose electrophoresis (1uL/lane)
and/or spectrophotometry (1:50 or 1:100 dilution). OD is less accurate due
to contaminating primer/free nucleotides. Gel shows whether there is
product or not. Do not procede with labeling if no product seen.
Ready for Cy3/5 dUTP labeling with random hexamer priming. Label at least 5
micrograms of amplified RNA per reaction. Use 6-8 micrograms of random
hexamers per labeling reaction.

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10X Second Strand Buffer:

200 mM Tris pH 6.9
900 mM KCl
46 mM MgCl2
1.5 mM Nicotine Adenine Dinucleotide (Calbiochem #481915)
100 mM (NH4)2SO4

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Linear Acrylamide Recipe
(from Joe Derisi, Brown lab)

for 375 uL of 10mg/ml stock:

75 uL 5% acrylamide:
3.75 mg acrylamide
1.5 uL 2M Tris pH 8
0.5 uL 3M NaAc
0.1 uL 0.5M EDTA
73 uL water

add 1 uL 10% ammonium persulfate and 0.1 uL TEMED
polymerize 30 minutes
add 2.5 volumes absolute etoh
spin 5 minutes top speed
aspirate supernatant
redissolve in 375 uL (DEPC) water for final concentration of 10 ug/uL

dilute 1:100 in DEPC water for use in Eberwine antisense RNA amplification
protocol

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References:

Luo, et al., Nature Medicine 5(1):117, 1999.
Carninci, et al., PNAS 95:520, 1998.
Eberwine, et al., PNAS 89:3010, 1992.
Van Gelder, et al., PNAS 87:1663, 1990.