TargetAmp™ 2-Round Biotin-aRNA Amplification Evaluation Kit

Catalogue number

Pack size

TAB2R71010-EV

10 Reactions

TargetAmp™ 1-Round Biotin-aRNA Amplification Evaluation Kit

Catalogue number

Pack size

TAB1R80510-EV

10 Reactions

TargetAmp™ 1-Round Biotin-aRNA Amplification Kit 105

Catalogue number

Pack size

TAB1R80510

10 Reactions

TAB1R80524

24 reactions

TargetAmp™ 2-Round Biotin-aRNA Amplification Kit

Catalogue number

Pack size

TAB2R71010

10 Reactions

TAB2R71024

24 Reactions

Epicentre® currently offers four TargetAmp™ aRNA Amplification Kits for the preparation of aRNA (cRNA). This high quality aRNA, after labelling, can be applied to virtually any DNA microarray platform including: Affymetrix GeneChip®, ABI, Agilent, GE CodeLink™, and other commercial chips or self-spotted arrays. TargetAmp produced aRNA can also be used for gene expression analysis using quantitative RT-PCR methods.

Selecting the optimal TargetAmp aRNA Amplification Kit for your experiments requires determination of the following before making a kit selection decision:

1. The quantity of total RNA available in your samples.
2. The total quantity of aRNA required for your application based on:
   1. The target requirements of your array platform.
   2. The number of replicates required (e.g., per condition) for statistical validity.
   3. The quantity of aRNA required for validation by qRT-PCR.
   4. Sample archiving requirements.
3. Your target labelling method.

Direct labelling with a biotinylated nucleotide is the current prevailing method due to introduction by Affymetrix early in the commercialization of microarray technology. However, an increasingly popular method incorporates aminoallyl-UTP into aRNA. The key advantages of aminoallyl labeling over direct labeling include higher yields, greater dye incorporation efficiency and lower background. Costs can also be significantly lower (by 30% to 50%) per reaction.

See the Epicentre's®TargetAmp Kit Performance web page for additional information, including yield, quality and data on the preservation of relative transcript abundance.

	1-Round Amplification		2-Round Amplification
	TargetAmp™ 1-Round Aminoallyl-aRNA Amplification Kit 101	TargetAmp™ 1-Round aRNA Amplification Kit 103	TargetAmp™ 2-Round Aminoallyl-aRNA Amplification Kit 1.0	TargetAmp™ 2-Round aRNA Amplification Kit 2.0
Starting Total RNA per Reaction	25ng - 500ng	25ng - 500ng	10pg - 500pg	10pg - 500pg
Typical Yield of Amplified aRNA	3µg - 60 µg	3µg - 60 µg	1.5µg - 70 µg	1.5µg - 70 µg
Reverse Transcriptase(s) Used	SuperScript™ III (provided by the user)	SuperScript™ III (provided by the user)	SuperScript™ III & SuperScript™ II (provided by the user)	SuperScript™ III & SuperScript™ II (provided by the user)
Fold Amplification	>5000	>5000	>5,000,000	>5,000,000
Time Required	6 hours	6 hours	2 days	2 days
End Product	Aminoallyl-aRNA	Unlabelled-aRNA	Aminoallyl-aRNA	Unlabelled-aRNA

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 (Aminoallyl-aRNA). High yields of aRNA (or aminoallyl-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) 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 (Aminoallyl-aRNA). 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.

Last modified: 2008-05-14 16:46:46

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Last modified: 2008-07-24 12:01:03

Protocols for: TargetAmp™ 1-Round Amplification Kits

TargetAmp™ 1-Round Aminoallyl-aRNA Amplification Kit 101 Protocol

(catalogue number TAA1R5605 / TAA1R4910 / TAA1R4924)

TargetAmp™ 1-Round aRNA Amplification Kit 103 Protocol

(catalogue number TAU1R5705 / TAU1R5110 / TAU1R5124)

TargetAmp™ 1-Round Biotin-aRNA Amplification Kit 105 Protocol

(catalogue number TAB1R6910 / TAB1R6924)

Protocols for: TargetAmp™ 2-Round Amplification Kits

TargetAmp™ 2-Round Aminoallyl-aRNA Amplification Kit 1.0 Protocol

(catalogue number TAA2R5605 / TAA2R4910 / TAA2R4924)

TargetAmp™ 2-Round aRNA Amplification Kit 2.0 Protocol

(catalogue number TAU2R5110 / TAU2R51224)

TargetAmp™ 2-Round Biotin-aRNA Amplification Kit 3.0 Protocol

(catalogue number TAB2R71010 / TAB2R71024)

TargetAmp™ Protocol Frequently Asked Questions

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-15 10:40:52