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The TargetAmp 1- & 2-Round Amplification Kits utilize a simplified and
improved "Eberwine" linear RNA amplification procedure, originally developed
in the laboratory of Professor James Eberwine (RN Van Gelder et. al., 1990, PNAS
USA 87:1663) at the University of Pennsylvania. The Eberwine RNA
amplification process is the most commonly used RNA amplification procedure
and is generally recognized as the method that provides the most reproducible
RNA amplification results.
The TargetAmp RNA Amplification Kits
utilize an improved and simplified Eberwine RNA Amplification process.
- The TargetAmp 1-Round amplification procedure can be completed in 6 hours.
- The TargetAmp 2-Round amplification procedure can be completed in 2 days.
- High temperature 1st-strand and 2nd-strand cDNA synthesis reactions ensure
the highest possible yields of full-length cDNA.
- No need to purify the cDNA prior to the in vitro transcription reaction.
- The TargetAmp Kits incorporate EPICENTRE's AmpliScribe™ high yield in
vitro transcription technology for the highest yield of aRNA or aminoallyl-aRNA.
- The TargetAmp Kits are optimized for use with SuperScript™ II and
SuperScript™ III Reverse Transcriptases (provided by the user).
- Combined reagents to minimize pipetting steps.
Overview of the TargetAmp™ 1-Round & 2-Round
RNA Amplification Processes
 1.
First-strand cDNA Synthesis. Poly(A) RNA of a total RNA sample is reverse
transcribed into first strand cDNA. The reaction is primed from an oligo(dT)
primer containing a phage T7 RNA Polymerase promoter sequence at its 5'-end.
First strand cDNA synthesis is catalyzed by SuperScript™ III Reverse
Transcriptase (provided by the user) and performed at an elevated temperature
to reduce RNA secondary structure.
2. Second-strand cDNA Synthesis. The RNA of the cDNA:RNA hybrid produced
in Step 1 is digested into small RNA fragments using an RNase H enzyme.
The RNA fragments then prime 2nd strand cDNA synthesis. The resulting double-stranded
cDNA contains a T7 transcription promoter in an orientation that will generate
anti-sense RNA (aRNA; also called cRNA) during the in vitro transcription
reaction. The cDNA produced can be used in the in vitro transcription
reaction without the need for purification.
3. In Vitro Transcription of aRNA or Aminoallyl-aRNA. High
yields of aRNA, aminoallyl-aRNA, or biotin-aRNA are produced in a rapid in
vitro transcription reaction that utilizes the double-stranded cDNA produced
in Step 2.
4. Purification of the aRNA (aminoallyl-aRNA) The aRNA, aminoallyl-aRNA,
or biotin-aRNA produced in Step 3 is purified by spin column chromatography
(supplied by the user).
The TargetAmp 1-round amplification procedure is now complete. The TargetAmp
2-round amplification procedure continues with steps 5-8.
5. Round Two, 1st-Strand cDNA Synthesis. The aRNA produced and purified
in the first round amplification process is reverse transcribed into first strand
cDNA using SuperScript II Reverse Transcriptase (Invitrogen; supplied by the
user). The reaction is primed using random sequence hexamer primers.
6. Round Two, 2nd-Strand cDNA Synthesis. The RNA of the cDNA:aRNA hybrid
produced in Step 5 is digested to small RNA fragments by RNase H. Second-strand
cDNA synthesis is then primed using a T7-Oligo(dT) Primer. The resulting product
is a double-stranded cDNA containing a T7 transcription promoter in an orientation
that that will generate aRNA during the second round in vitro transcription
reaction.
7. In Vitro Transcription of aRNA (AminoallylaRNA). High
yields of aRNA (or aminoallyl-aRNA) are produced in an in vitro transcription
reaction that utilizes the double-stranded cDNA produced in Step 6.
8. Purification of the aRNA (aminoallyl-aRNA) The aRNA (aminoallyl-aRNA)
produced in Step 7 is purified by spin column chromatography (supplied by the
user).
The TargetAmp 2-Round amplification procedure is now complete.
A TargetAmp 1-Round RNA amplification reaction
can be completed in 6 hours.
A TargetAmp 2-Round RNA amplification reaction can be completed in 2
days.
| |
Round 1
1st-Strand cDNA synthesis |
35 minutes |
35 minutes |
Round 1
2nd-Strand cDNA synthesis |
13 minutes |
26 minutes |
Round 1
In vitro transcription |
4 hours, 15 minutes |
4 hours, 15 minutes |
| aRNA, aminoallyl-aRNA, or biotin-aRNA purification |
About 30 minutes |
About 30 minutes |
Round 2
1st-Strand cDNA synthesis |
|
1 hour, 37 minutes |
Round 2
2nd-Strand cDNA synthesis |
|
28 minutes |
Round 2
In vitro transcription |
|
9 hours, 15 minutes |
| aRNA or aminoallyl-aRNA purification |
|
About 30 minutes |
| TOTAL TIME |
5 hours, 30 minutes |
17 hours, 30 minutes |
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