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Efficient RNA Labeling with AF (Alexa Fluor®)-Dyes

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So far in vitro transcription-based direct labeling of RNA probes with AF (Alexa Fluor®)-Dyes is rather inefficient.

We now offer HighYield T7 RNA Labeling Kits for efficient AF (Alexa Fluor®)-Dye labeling.

AF (Alexa Fluor®)-Dyes are hydrophilic fluorescent dyes with remarkable photostability however, when conjugated to normal 5-UTP, they show rather poor incorporation (Fig. 1, right panel).

In contrast, when the dye is conjugated via a linker-optimized UTP (here PEG5), efficient preparation of dye labeled RNA probes (> 20 nt up to several thousand nt) is easily achieved (Fig. 1, left panel; Tab. 1).

Figure 1: Efficient RNA Labeling with HighYield T7 AF488 RNA Labeling Kit.

Figure 1: Compared to non-linker AlexaFluor488-UTP, linker-optimized UTP-PEG5-AF488 (in combination with an optimized enzyme mix) shows increased labeling efficiency (A) and product yield (B), the optimum is observed with 40% UTP-substitution.

A) Visualization of labeled RNA without RNA intercalator. B) Visualization of total RNA with RNA intercalator EtBr. 0 – 100 %: Increasing percentage of UTP substitution by labeled UTP.

1.4 kbp RNA was generated using HighYield T7 AFXXX RNA Labeling Kit components in combination with UTP-PEG5-AF488 or Alexa Fluor®488-5-UTP (Thermo Scientific). Reaction conditions: 1 µg DNA template, 30 min at 37°C. Crude in vitro reaction mix was subsequently analyzed be agarose gel electrophoresis.

Table 1: Overview on available HighYield T7 AFXXX (Alexa Fluor®XXX) RNA Labeling Kits

Product Dye Substitute for Excmax/ Emmax
Recommended range of substitution Average yield*
HighYield T7 AF488 RNA Labeling Kit AF488 (also known as Alexa Fluor®488) Cy2 / FITC (Fluorescein) ATTO488 494/517 30 - 50 % 5 - 7 µg
HighYield T7 AF555 RNA Labeling Kit AF555 (structural analog to Alexa Fluor®555) Cy3 / ATTO550 555/572 30 - 50 % 5 - 7 µg
HighYield T7 AF594 RNA Labeling Kit AF594 (also known as Alexa Fluor®594) Texas Red 590/617 30 - 50 % 5 - 7 µg
HighYield T7 AF647 RNA Labeling Kit AF647 (structural analog to Alexa Fluor®647) Cy5 / ATTO647N 648/671 20 - 40 % 4 - 6 µg
* 20 µl reaction after 30 min at 37°C (1 µg T7 G-initiating control template, 1.4 kbp RNA transcript, 35% UTP-PEG5-AFXXX)
Dr. Barbara Zschoernig

Please contact Barbara with all questions or inquiries you may have!