Fluorescence dye Cy5.5-based primer extension technique determines RNA cleavage sites of bacteria

Abstract:

[Background] Traditional primer extension assay usually uses radioactive probes to identify the start sites of transcription and processing sites of transcripts. However, the application of this method in general laboratories is limited because of the short half-life, health hazards, and waste disposal issue of radioisotope-labeled primers. [Objective] To develop a non-radioactive method of primer extension by employing near-infrared fluorescent cyanine dye Cy5.5 and then employ this method to identify the RNA cleavage sites of bacteria. [Methods] The sequence harboring RNA cleavage sites was inserted into the dual-luciferase reporter assay system, and then the Cy5.5-labeled oligonucleotides primer specific to mcherry was used for primer extension. The RNA cleavage sites were precisely identified by Northern blotting and Cy5.5-based primer extension technique. [Results] Two processing sites of cip-cel mRNA were identified, which located in the downstream region of the stem-loop. However, no conserved sequence was identified near the processing sites. [Conclusion] Compared with radioactive isotope-based primer extension methods, the Cy5.5-based primer extension technique not only efficiently determines processing sites of RNA molecules but also avoids the use of hazardous radioactive isotope reagents for labeling and shortens the processing time, which suggests that the Cy5.5-labeled probe is suitable for primer extension assay.