How does one sequence a RNA hairpin structure?

09 March 2025 1 705 Report

I am having trouble sequencing RNA due to a hairpin structure in the middle of my sequence. I have the bordering regions but there's still a gap, probably due to the polymerase falling off. Is there any way to prepare the RNA differently, perhaps denature the secondary structure before moving on to cDNA synthesis?

Margaret Bohmer

A simple heat denaturation step should work. Calculate the Tm of your RNA (there are multiple online calculators for this). Heat your samples for ~5 min in the thermocycler at a temperature above the Tm, then put on ice.

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