Navigation Links
Amino Allyl Labeling for Array Analysis


RNA is often labeled with fluorescent dyes (i.e. Cy3 or Cy5) for microarray analysis. Label may be directly incorporated during cDNA synthesis, aRNA amplification or a post-aRNA reverse transcription (RT) reaction, or attached via a chemical coupling reaction performed after any enzymatic reactions (post-synthesis). Direct labeling requires the enzymatic incorporation of a Cy-labeled dNTP/NTP into the cDNA, aRNA or RT product. Alternatively, indirect labeling usually incorporates an intermediary nucleotide to which the fluorescent label is later attached. Although seemingly more convenient, direct labeling can be problematic for a number of reasons. Cy-labeled dNTPs/NTPs are not incorporated by reverse transcriptase or RNA polymerases with the same efficiency as unlabeled NTPs, leading to variable incorporation efficiency of different dyes (e.g. Cy3-NTP vs. Cy5-NTP). In addition, to be effective, direct incorporation often requires the use of more expensive modified nucleotides.

Post-synthesis labeling avoids the problems inherent to direct enzymatic incorporation of Cy-labeled dNTPs/NTPs. With indirect labeling, amine-modified NTPs (amino allyl-dUTP/UTP) are incorporated during reverse transcription or aRNA amplification. Amino allyl-NTPs are incorporated with similar efficiency as unmodified NTPs during enzymatic reactions such as reverse transcription. Coupling of Cy3 or Cy5 dyes to the amino allyl cDNA or aRNA, post-synthesis, results in probe populations with equivalent labeling efficiencies.

Amino allyl-dUTP contains a reactive amino group on a 2-carbon spacer attached to the methyl group on the base portion of dUTP. During the coupling reaction, after cDNA or aRNA synthesis, this amino group reacts with the NHS ester of a monoreactive Cy dye.

Ambion has several products for the post-synthesis labeling of nucleic acids for array analysis. The Amino Allyl cDNA Labeling Kit contains all the necessary reagents (except monoreactive Cy dyes) for the synthesis of amino allyl labeled cDNA by reverse transcription and subsequent Cy dye coupling. The MessageAmp aRNA Kit generates amplified aRNA for array analysis. Amino allyl UTP can be incorporated into the transcription step of this procedure to produce amino allyl labeled RNA ready for Cy dye coupling. Finally, amino allyl dUTP/UTP, available separately from Ambion, can be incorporated into any reverse transcription reaction.


Cy3 and Cy5 monoreactive dyes are available for Amersham Biosciences.
Cy3 and Cy5 are trademarks of Amersham Biosciences.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
2. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
3. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
4. New Microarray Labeling Kit for Preparing cDNA Probes
5. aRNA Synthesis and Labeling for Array Analysis
6. Fluorescently Labeling Long dsRNA with the Silencer siRNA Labeling Kit
7. Efficient Europium Labeling of Lymphokine-Activated Killer Cells Using the Gene Pulser II with RF Module, Rev C
8. USB OptiKinase Eliminates Base Bias in Labeling of Oligonucleotide 5-Ends
9. Innovative Tissue Array Technology for High-Throughput Screening of Gene Expression
10. Human Universal cDNA Library Array I
11. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/24/2017)... ... ... Delpor, Inc. (Delpor), a biotechnology company focused on drug delivery, today announced ... for the further advancement of the company’s 3-month olanzapine product ( DLP-119 ). The ... therapeutic levels of olanzapine for a period of 3 months., “We are honored and ...
(Date:2/24/2017)... , Feb. 24, 2017 Symic Bio, ... developing a new category of therapeutics, announced today the ... SB-030 in peripheral artery disease. The trial will evaluate ... single-use therapeutic, in the reduction of restenosis following angioplasty. ... critical development milestone for SB-030," said Nathan Bachtell ...
(Date:2/24/2017)... Inc. (NASDAQ: CBPO) ("China Biologic" or the "Company"), a leading ... financial results for the fourth quarter and fiscal year of ... Total sales in the fourth quarter of ... 13.6% in USD terms to $77.6 million from $68.3 million ... profit increased by 13.3% to $46.8 million from $41.3 million ...
(Date:2/23/2017)... 2017 ... share data, unaudited)Three Months Ended December 31,Twelve Months Ended ... $           ... 89026%Aldurazyme Net Product Revenue 3539(10)%9498(4)%Kuvan ... Product Revenue  756025%297303(2)%Vimizim Net Product ...
Breaking Biology Technology:
(Date:2/7/2017)... , February 7, 2017 Ipsidy ... Solutions Corporation [OTC: IDGS], ("Ipsidy" or the "Company") a ... transaction processing services, is pleased to announce the following ... Effective January 31, 2017, Philip D. ... Directors, CEO and President.  An experienced payment industry professional ...
(Date:2/2/2017)... NEW YORK , Feb. 2, 2017  EyeLock ... has released a new white paper " What You ... The problem of ensuring user authenticity is a ... protect the authentication of users. However, traditional authentication schemes ... Biometric authentication offers an elegant ...
(Date:1/26/2017)... CITY , Jan. 26, 2017  Crossmatch, a ... unveiled a new solution aimed at combatting fraud, waste ... solution was introduced at the Action on Disaster Relief ... key meeting point for UN agencies and foreign assistance ... Fraud, waste and abuse are a largely unacknowledged ...
Breaking Biology News(10 mins):