What is the advantage of producing aminoallyl-aRNA rather than direct incorporation of a biotin-NTP, Cy-NTP or other fluorescent dye-NTP into the in vitro transcription reaction?
The aminoallyl-method for indirect labeling of the target nucleic acid is becoming increasingly popular because it has important advantages over direct incorporation of a biotin-NTP or fluorescent-NTP, including: • A single RNA amplification reaction, producing aminoallyl-aRNA, can be divided and labeled independently with different NHS-ester (e.g. Cy3-NHS and Cy5-NHS) if desired. • An aminoallyl-NTP is more efficiently incorporated into the aRNA during the in vitro transcription reaction resulting in higher yields of aminoallyl-aRNA than can be obtained by direct incorporation of a biotin-NTP or a fluorescent-NTP. • Conjugation of an amine-reactive biotin-NHS, Cy-NHS or other fluorescent dye-NHS to AA-aRNA is a much less expensive way to label the target compared to direct incorporation of a biotin-NTP, Cy-NTP or fluorescent dye-NTP. • Inexpensive Biotin-X-X-NHS, Cy-NHS and fluorescent dye-NHS labeling reagents are commercially available.
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