TargetAmp™ aRNA Amplification Process

TargetAmp™: Selection Guide | Process | Performance | FAQ | Poster (3 MB PDF)

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.

  TargetAmp 1-Round Kits TargetAmp 2-Round Kits
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