TargetAmp 1-Round aRNA Amplification Kit 103
RNA Amplification Procedure
A.
B.
C.
D.
First-strand cDNA Synthesis
Second-strand cDNA Synthesis
In Vitro Transcription of aRNA
aRNA Purification
A. First-strand cDNA Synthesis
Required for Step A:
TargetAmp T7-Oligo (dT) Primer A
TargetAmp Reverse Transcriptase PreMix-SS
Dithiothreitol (DTT)
RNase-Free Water
Superscript III Reverse Transcriptase (200 U/μl)
(Red Tube)
(Red Tube)
(Colorless Tube)
(Colorless Tube)
(Not Provided in Kit)
Incubations Performed in Step A: 50 ºC and 65 ºC. If the thermocycler is used make sure the
lid function is also set at 50 ºC and 65 ºC in each incubation step.
1. Anneal the TargetAmp T7-Oligo (dT) Primer A to the RNA sample. For best results use no
more than 500 ng of RNA for each reaction. Perform the reaction in 0.2 mL tubes (this will
reduce evaporation during incubation).
x μl RNase-Free Water
x μl Total RNA sample (500ng)
1 μl TargetAmp T7-Oligo (dT) Primer A
3 μl Total reaction volume
2.
3.
4.
5.
Gently mix the reaction by flicking and spin down the contents before incubating.
Incubate at 65 ºC for 5 minutes.
Chill on ice for 1 minute
Prepare the 1st-strand cDNA Synthesis Master Mix
For each 1st-strand cDNA synthesis reaction, combine on ice:
1.50 μl TargetAmp Reverse Transcription PreMix-SS
0.25 μl DTT
0.25 μl Superscript III Reverse Transcriptase (200 U/μl)
2.00 μl Total reaction volume
6. Gently mix the 1st-strand cDNA Synthesis Master Mix and add 2 μl to each reaction.
7. Gently mix the reactions and incubate at 50 ºC for 30 minutes.
B. Second-strand cDNA Synthesis
Required for Step B:
TargetAmp DNA Polymerase PreMix-SS 1
TargetAmp DNA Polymerase SS-1
(Red Tube)
(Red Tube)
Incubations Performed in Step B: 65 ºC and 80 ºC.
1. Prepare the 2nd-Strand cDNA Synthesis Master Mix.
For each 2nd-strand cDNA synthesis reaction, combine on ice:
4.5 μl TargetAmp DNA Polymerase PreMix-SS 1
0.5 μl TargetAmp DNA Polymerase
5.0 μl Total reaction volume
2.
3.
4.
5.
6.
Gently mix the 2nd-Strand cDNA Synthesis Master Mix and add 5 μl to each reaction.
Gently mix the reactions and incubate at 65 ºC for 10 minutes.
Centrifuge down the contents.
Incubate at 80 ºC for 3 minutes.
Chill on ice
Note: If desired, the reactions may be frozen and stored overnight at -20 ºC.
C. In Vitro Transcription of aRNA
Required for Step C:
TargetAmp T7 RNA Polymerase
TargetAmp T7 Transcription Buffer
100 mM ATP
100 mM CTP
100 mM GTP
100 mM UTP
Dithiothreitol
RNase-Free Water
RNase-Free DNase I
(Green Tube)
(Green Tube)
(Green Tube)
(Green Tube)
(Green Tube)
(Green Tube)
(Colorless Tube)
(Colorless Tube)
(Green Tube)
Incubations Performed in Step A: 37 ºC and 40 ºC
1. Warm the TargetAmp T7 RNA Polymerase to room temperature.
2. Warm the TargetAmp T7 Transcription Buffer and RNase-Free water at 37 ºC for 5 minutes.
Mix the buffer thoroughly to dissolve any precipitates and keep it at room temperature.
3. Prepare the In Vitro Transcription Master Mix.
Important: Add reagents in order. Mix RNase-Free Water and TargetAmp T7
Transcription Buffer before adding the rest of the reagents.
For each in vitro transcription reaction, combine at room temperature:
13.6 μl RNase-Free Water
4.0 μl TargetAmp T7 Transcription Buffer
3.6 μl ATP
3.6 μl CTP
3.6 μl GTP
3.6 μl UTP
4.0 μl DTT
4.0 μl TargetAmp T7 RNA Polymerase
40.0 μl Total reaction Volume
4. Gently mix the In Vitro Transcription Master Mix and add 40 μl to each reaction.
5. Gently mix the reactions and incubate at 42 ºC for 4 hours. For optimal yield and quality do
not exceed 4 hours of incubation.
6. Add 2 μl of RNase-Free DNase I to each reaction.
7. Gently mix and incubate at 37 ºC for 15 minutes.
D. aRNA Purification
Required for Step D:
Qiagen RNeasy Mini Kit
RNase-Free Water (use the water provided by the Qiagen RNeasy Mini Kit)
RLT/β-Mercaptoethanol Solution (RLT provided in kit, β-ME not provided in kit)
RPE Solution (provided in kit)
100% Ethanol
80% Ethanol
1.7 mL centrifuge tubes
Pink spin columns (provided in kit)
1. For each sample prepare 350 μl RLT/β-ME Solution. Make the solution under the fume hood.
To 1.0 mL Buffer RLT add 10 μl of β-ME.
2. For each sample prepare 650 μl RPE Solution. The RPE Solution in the kit should be diluted
1 volume RPE to 4 volumes 100% Ethanol. Check to see if the solution bottle is marked as
already being diluted 1:4. If you have to dilute a new solution, make sure you mark the bottle
as being diluted.
3. Transfer each sample to 1.7 mL centrifuge tubes.
4. To each sample add:
48 μl RNase-Free Water
350 μl RLT/β-ME Solution
250 μl 100%
5. Apply each sample to the pink spin column in a 2 mL collection tube (provided in kit).
 Centrifuge at >8,000 x g for 20 seconds.
 Discard the flow-through
6. Apply 650 μl RPE Solution onto each column.
 Centrifuge at >8,000 x g for 20 seconds.
 Discard the flow-through
7. Apply 650 μl 80% Ethanol onto each column.
 Centrifuge at >8,000 x g for 20 seconds.
 Discard the flow-through
8. Transfer the column to a new collection tube (provided in kit).
 Centrifuge at max speed for 5 minutes.
 Keep the spin column and discard the collection tube.
9. Transfer the spin column to a 1.7 mL centrifuge tube.
10. Apply 25 μl RNase-Free Water directly onto the center of the silica-gel membrane of the spin
column.
 Wait for 2 minutes.
 Centrifuge at full speed for 1 minute.
11. Repeat step 10 in the same 1.7 mL collection tube.
12. The reactions can now be stored at -80 ºC.