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: 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.
| Product | Dye | Substitute for | Excmax/ Emmax [nm]* |
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 |
HighYield T7 RNA Labeling Kits for Biotin- or Digoxigenin modification are available as well!
Please contact Barbara with all questions or inquiries you may have!
