Tuesday, April 27, 2010

Labeling RNA for gene expression analysis

Here are answers to questions that we commonly receive regarding the TargetAmp™ Antisense RNA (aRNA) Amplification Kits.

Can I use the TargetAmp Kits for producing fluorescent-labeled aRNA?
Yes. The TargetAmp 1-Round and the TargetAmp 2-Round Aminoallyl-aRNA Amplification Kits produce aminoallyl-aRNA (AA-aRNA), which can be readily labeled with a fluorescent dye conjugated to N-hydroxysuccinimide (NHS). 

What are the advantages of aminoallyl-based (indirect) labeling over direct incorporation of a labeled NTP?
Aminoallyl-UTP is more efficiently incorporated into the aRNA during the in vitro transcription reaction, compared to labeled nucleotides. Additionally, the conjugation of an amine-reactive NHS ester of biotin or a fluorescent dye to AA-aRNA can be a less expensive method to label the target compared to direct incorporation of labeled nucleotides. 

What are the advantages of direct labeling of aRNA?
Labeling by direct incorporation is faster compared to the aminoallyl-based procedure. Conjugation of AA-aRNA to a dye- or biotin-NHS ester after in vitro transcription requires a 1-hour incubation, and an additional clean-up step, compared to direct incorporation. Further, labeling aRNA by direct incorporation does not require the use of toxic reagents (such as DMSO), which are used in the indirect labeling protocol.

To find the TargetAmp Kit that’s right for your application, please see our selection guide.

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