hello guys. I am working on noncoding rna (short & long both).
which method is better to build secondary structure of ncRNA?
what criteria should be taken for precursor sequence up & downstream both (in case of plants). shall it same for all plants.
After build hypothetical structure through software how to validate these structures.
For in silico long noncoding RNA folding I typically use the Vienna package (http://rna.tbi.univie.ac.at/). If you want as robust of a folding prediction as possible, I recommend sequencing the 5' and 3' ends of your RNA using RACE. To validate RNA structure, old school structural probing via single strand and double strand RNAses is typically the way to go - at least if you are looking at just a couple of RNAs. Typically you digest a RNA that has radioactive nucleotides incorporated. Running the digested RNAs out on an acrylamide gel and looking for different patterns of digestion is pretty straightforward. However, it is highly dependent on having optimal folding conditions (as this is all in vitro).
If you are looking at a bunch of RNAs, there are more high-throughput ways that are either in vitro or in vivo, but they can be a bit more expensive.
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