Ambion’s MessageAmp™ aRNA Kit: RNA Amplification for Array Analysis
(Lab of Klaus Kaestner, UPenn, Philadelphia, PA)
Ambion Catalog # 1750, 20 rxns, $795.00
*** Ambion recommends using the smallest tubes possible for all steps to minimize
evaporation. Labs that can accommodate the heating/cooling of smaller tubes (ie, PCR
tubes, 200  tubes, etc.) should use the smallest size(s) at all steps possible. We recommend
processing no more than 6 samples plus one Ambion Kit positive control per procedure.
These sample instructions are written for 6 RNA samples plus 1 Ambion Kit positive control
sample using 1.5 ml eppendorf tubes and standard heating/cooling blocks. All RNA
handling precautions apply throughout the procedure.
Day #1:
First-Strand cDNA Synthesis:
1.
Set heating/cooling blocks to the following temperatures:
70°C
42°C
16°C
50°C
An incubator at 37°C is also needed.
2.
Thaw RNA samples from –80°C freezer storage.
3.
Label 1.5 ml tubes for amplification. It is recommended that no more than 6
samples plus one AMBION kit positive control sample be used in one procedure.
4.
Aliquot ~ 200 ng RNA in tube #1-6 and 1000 ng Ambion Control RNA (1 @ 1
ug/) in tube #7 in a volume of NO MORE than 11.
5.
Add 1  of T7 Oligo(dT) Primer to each tube (Ambion Kit).
6.
Add Nuclease-free water (Ambion Kit) to a final volume of 12 . [This volume
includes the RNA, Nuclease-free water, and Oligo(dT) primer.]
7.
Pulse (briefly centrifuge) samples and then incubate at 70°C for 10 minutes.
After 10 minutes, pulse samples and put at 42°C while making up Reverse
Transcription Master Mix (next step).
8.
Make Reverse Transcription Master Mix (Ambion Kit components) in a 1.5 ml
eppendorf tube:
10X First Strand Buffer
Ribonuclease Inhibitor
dNTP Mix
x1
2
1
4
x8 (need enough for 7)
16 
8
32 
9.
Flick tube to mix, pulse, and incubate @ 42°C for 2 minutes.
10.
Add 7  Reverse Transcription Master Mix to each RNA sample tube.
11.
Add 1  Reverse Transcriptase enzyme (Ambion Kit) to each tube. Mix and
pulse samples.
12.
Incubate for 2 hours at 42°C.
13.
After 2 hours, pulse and store samples on ice until second strand cDNA synthesis
reaction (below).
Second-Strand cDNA Synthesis:
1.
Make Second-Strand Synthesis Mix (Ambion Kit components) at room
temperature:
Nuclease-free water
10X Second Strand Buffer
dNTP mix
DNA Polymerase
RNase H
x1
63 
10 
4
2
1
x8 (need enough for 7)
504 
80 
32 
16 
8
2.
Mix contents well, pulse, and add 80 ul Second-Strand Synthesis Mix to each of
the 7 samples. Mix and pulse.
3.
Incubate for 2 hours at 16°C. After 2 hours, pulse samples and proceed directly to
the next cDNA purification steps.
cDNA Purification:
1.
Aliquot Nuclease-free water into a 1.5 ml eppendorf tube. You will need 100 
per sample. 7 samples = 700 . Aliquot 800  and place in 50°C heat block.
2.
Place Ambion Kit Filter Cartridge in the supplied 2 ml (labeled!) eppendorf tube.
Do not label filter cartridge … writing comes off during washes.
3.
Add 100  cDNA Binding Buffer (Ambion Kit, stored @ 4°C) to empty Filter
Cartridges that you placed on supplied 2-ml eppendorf tubes in step 2. **Make
sure buffer is not precipitated in the bottle … you may need to run bottle under
hot water before use to re-dissolve.
4.
Incubate at room temperature for 5 minutes to equilibrate Filter Cartridge.
5.
Add 250  cDNA Binding Buffer to each cDNA sample (NOT to Filter
Cartridge). Mix thoroughly.
6.
Pipet cDNA sample/cDNA Binding Buffer mixture onto equilibrated Filter
Cartridge.
7.
Close caps and centrifuge for 1 minute @ 8000 rpm (10000 g) or until mixture
has passed through the filter.
8.
Discard liquid flow-through and replace the same Filter Cartridge back onto the
same labeled 2-ml tube.
9.
*Make sure EtOH has been added to the bottle of cDNA Wash Buffer (11.2 ml,
ACS grade, 100% EtOH).
Apply 650  cDNA Wash Buffer (Ambion Kit, stored @ 4°C) to each Filter
Cartridge.
10.
Close caps and centrifuge for 1 minute @ 8000 rpm (10000 g) or until buffer
passes through the filter.
11.
Discard liquid flow-through. Replace Filter Cartridge onto same 2-ml tube.
12.
Close caps and spin 1 minute @ 8000 rpm (10000 g) to remove trace amounts of
EtOH.
13.
Transfer Filter Cartridges to fresh labeled 2 ml tubes (supplied).
14.
Apply 50  pre-heated (50°C) Nuclease-free water to each Filter Cartridge.
15.
Let water stand on filter for 2 minutes.
16.
Close caps and spin for 1 minute at 8000 rpm (10000 g) until all water passes
through filter. Don’t touch flow-through! Leave it there!
17.
Apply another 50  pre-heated (50°C) Nuclease-free water to Filter Cartridge.
18.
Let water stand on filter for 2 minutes.
19.
Close caps and spin for 1 minute @ 8000 rpm (10000 g) or until all water passes
through filter. You now have 100  eluted double-stranded cDNA in the 2 ml
tube.
20.
Discard the filter cartridge.
Concentrate the purified double-stranded cDNA:
1.
Spin tubes with caps open (balance!) in vacuum centrifuge, high heat.
2.
Check samples every 5-10 minutes until it reaches a volume of 20 . Then, check
every 1-2 minutes until it reaches a volume of <8 . You may want to use
eppendorf tubes filled with 20  and 8  of water for easy visual comparison of
samples.
3.
Remove samples from speed-vac when volume reaches <8 . Turn off speed-vac.
4.
Adjust volume to 8  with Nuclease-free water. To do this, pipet existing volume
with P10 pipetman set for 10  … turn volume dial down until liquid reaches the
end of the pipet tip to see current volume. Subtract from 8  and make up the
difference with Nuclease-free water.
In Vitro Transcription to Synthesize aRNA (Unmodified aRNA Synthesis):
1.
Make In Vitro Transcription Mix (Ambion Kit components):
T7 ATP Solution (75 mM)
T7 CTP Solution (75 mM)
T7 GTP Solution (75 mM)
T7 UTP Solution (75 mM)
T7 10x Reaction Buffer
T7 Enzyme Mix
x1
2
2
2
2
2
2
x 8 (need enough for 7)
16 
16 
16 
16 
16 
16 
** Make sure all components (except T7 Enzyme Mix) are at room temperature
before use to avoid unwanted precipitation of DNA.
2.
Add 12  In Vitro Transcription Mix to 8  cDNA samples. Mix well, pulse.
3.
Incubate reactions at 37°C overnight (An incubator at 37°C is best to minimize
evaporation). We find that 16-18 hours is appropriate.
Day #2:
DNase Treatment (to remove DNA template from aRNA):
1.
Pre-heat Nuclease-free water at 50°C (you will need 100  x 7 samples = 700 .
Aliquot 800  in eppendorf and place on benchtop heat block).
2.
Add 2  DNase I (Ambion Kit) to each reaction. Mix tubes, pulse.
3.
Incubate at 37°C for 30 minutes.
4.
Place one filter cartridge in a supplied labeled 2-ml tube. Do not label cartridge
because ink comes off with wash buffer.
5.
Add 100  aRNA Binding Buffer (Ambion Kit, stored @ 4°C) to top of cartridge.
6.
Incubate at room temperature for 5 minutes to equilibrate cartridge.
7.
Add 78  aRNA Elution Solution (Ambion Kit, stored @ 4°C) to each aRNA
sample. Final volume is now 100 .
8.
Mix thoroughly.
9.
Add 350  of aRNA Binding Buffer (Ambion Kit, stored @ 4°C) to each sample
and mix thoroughly.
10.
Add 250  ACS grade 100% EtOH (not supplied) to each sample. Mix
thoroughly.
11.
Pipet sample/binding buffer/EtOH mixture onto equilibrated cartridge.
12.
Centrifuge 1 minute @ 8000 rpm (10000 g) until mixture has passed through
filter.
13.
Discard liquid flow-through.
14.
Replace same filter cartridge on same 2-ml tube.
15.
*Make sure that EtOH has been added to aRNA Wash Buffer before using (20 ml,
ACS grade, 100% EtOH).
Apply 650  aRNA Wash Buffer (Ambion Kit, stored@ 4°C) to the filter
cartridge.
16.
Centrifuge for 1 minute @ 8000 rpm (10000 g) until wash solution passes
through.
17.
Discard liquid flow-through. Replace filter onto same 2-ml tube.
18.
Spin for 1 more minute @ 8000 rpm (10000 g) to remove trace amounts of EtOH.
19.
Transfer filter cartridge to a fresh labeled supplied 2-ml tube.
20.
Add 50  pre-heated (50°C) Nuclease-free water to the center of the cartridge.
21.
Leave at room temperature for 2 minutes.
22.
Centrifuge for 1 minute @ 8000 rpm (10000 g) until water passes through. Don’t
touch flow-through!!
23.
Add 50  more of pre-heated (50°C) Nuclease-free water to the cartridge.
24.
Centrifuge for 1 minute @ 8000 rpm (10000 g) until water passes through. aRNA
is now eluted in 100  Nuclease-free water.
25.
Discard filter cartridge.
Results/ Expected Yields:
We have found that the MessageAmp aRNA Kit produces anywhere from 100 to 1000fold amplification of mRNA starting material. From 200 ng of total RNA (~10 ng
mRNA), we have been able to attain 1-10 ug of amplified mRNA.
Detection of aRNA:
Visualization of aRNA on a 1% Agarose Gel using
SYBR GOLD from Molecular Probes:
SYBR Gold nucleic acid gel stain
Molecular Probes, S-11494
1.
2.
3.
Make 1% agarose gel with NO ethidium bromide using 0.5X TBE. DEPC water
is not necessary.
Aliquot 10  of aRNA (from total of 100  final elution) into new 1.5 ml
eppendorf tubes. Don’t forget to include the Ambion Kit positive control aRNA
as a sample to run on the gel.
Add 10  UREA Denaturing Loading Buffer to each sample. Mix.
UREA Denaturing Loading Buffer (stored @ -20°C)
2X TBE (pH 8.3)
13% Ficoll (w/v)
0.01% Bromophenol Blue
7M UREA
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Place samples with UREA Denaturing Loading Buffer at 65°C for 10 minutes.
Cool on ice for 1 minute.
Load 0.5  1 KB DNA ladder (Invitrogen, 15615-024). We use 0.5  of ladder
that has been diluted 1:10 with 1M TrisCl, 0.5M EDTA, H20, and DNA sample
loading dye (Ficoll, bromophenol blue).
Load all 20  of RNA/buffer samples (including positive control for comparison).
Run gel at ~ 120 mV in 0.5x TBE.
Make 1:10,000 dilution of SYBR GOLD in 0.5X TBE. For 200 ml, use 20 ul
SYBR GOLD (stored @ -20°C) in 200 ml 0.5X TBE. Keep diluted SYBR
GOLD in a flask wrapped in foil @4°C when not in use.
Once samples have run 1-2 inches from the wells, cut the portion of the gel that is
needed for visualization with a clean razor blade.
Put gel slab in an empty pipet tip box with a light-tight lid. Lab tape convering all
translucent parts of the tip box works well.
Pour in diluted SYBR GOLD. Cover unused solution with foil to keep light out.
Soak gel for >30 minutes with gentle rocking in the dark.
Save diluted SYBR GOLD in foil-wrapped flask @ 4°C. We find that a 1:10000
dilution is reusable and will be effective for at least one week.
Wash gel with 0.5X TBE by putting ~ 200 ml in pipet tip box and soaking for 5
minutes in the dark.
Photograph with gel-doc. You should see a smear that spans about 0-1600 bp.
Quantitation:
We use the RNA 6000 Nano Assay from Agilent Technology in conjunction with
the Agilent 2100 BioAnalyzer to quantitate our aRNA. It requires 1  of aRNA
sample (as long as the sample is between 5 and 100 ug/) to achieve accurate
quantitation. We follow the precise instructions included with the equipment and
the assay guide with no additions or alterations.