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Go to the RNAhybrid submission page at http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html.
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Paste the miRNA sequences in Fasta format into the miRNA sequence field. Alternatively you can upload a file that contains the sequences. In Fasta format, each sequence entry consists of a line beginning with the symbol '>' (the description line) and followed by the sequence's identifier and description, and one or more lines with the sequence itself. A Fasta file can contain multiple such sequence entries, in which case it is also called a multiple Fasta file. The sequence entries in such a file just follow each other, each one starting with a new line. For RNAhybrid, each entry has to have a unique identifier, and there must be no blanks between the '>' and the identifier.
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Paste your potential target sequences (eg. 3'UTRs) in Fasta format into the
target sequence field. Alternatively you can upload a file that contains
the sequences.
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If you would like to get an email notification once the calculations have
finished, give your email address in the respective field.
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Press the Submit button. After a short while the web server will get back
to you with the results.
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If you are unsure about what data to paste into the form, press the Example
button. This will insert example target and miRNA sequences into the
appropriate fields. Then press Submit and wait for the results.
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Before you submit, you may choose among the following options (just go back
from the results page with the browser's back-button):
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You can force RNAhybrid to consider only those target sites that fulfill
the seed criterion (Lewis et al., 2005), i.e. that have no unpaired
nucleotides in the seed region of the miRNA or in the corresponding seed-
match region of the target. A useful choice is to force a seed from
positions 2 to 7. If you want to be more restrictive, you can disallow any
non-Watson-Crick base pairing (G:U pairs) in the seed. These options will
reduce the number of spurious target sites, though at the risk of missing
bona-fide sites that do not have a seed. For plant miRNA target prediction
the seed region might be set from 8 to 12, so that the expected cleavage
site is covered.
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For the prediction of plant miRNA targets, where only few nucleotides in
the miRNA are unpaired, the options for restricting loop sizes are
especially useful. A useful choice might be 1 unpaired nucleotide on each
side of an internal loop and 1 unpaired nucleotide in a bulge loop.
Internal loops are regions in a hybridization duplex that consist of two
base pairs that enclose unpaired nucleotides on both sides, i.e. in the
target and in the miRNA. Bulge loops are similar, but have unpaired
nucleotides on only one side.
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If you have chosen to restrict your search to target sites that contain a
seed match, you can speed up the calculation by using the seed-match speed-
up option. With that, RNAhybrid first searches for seed-matches, and only
upon the detection of such a match performs the complete calculation of
optimal hybridization in a window around the seed match. A window size of
50 nt is suggested. For seeds of length 6, with no G:U pairs allowed, this
option accelerates the search by a factor of 8.
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For statistical guidance, i.e. p-value calculation, choose the "set"
option. In this option you can tell RNAhybrid from which organism your
target candidate sequences are. This information is used by RNAhybrid to
heuristically calculate p-values on the basis of the miRNA's "energy
potential". The larger this potential, the better chance hybridisations
will be, and p-values for actual binding energies are adjusted accordingly.
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You may also want to install RNAhybrid locally on your computer
system. Especially, if you would like to get p-values while using any of
the options 7.1 to 7.3, since the heuristic p-value calculation assumes
that none of these options is chosen. Follow the "download" link on the
RNAhybrid welcome page http://bibiserv.techfak.uni-bielefeld.de/rnahybrid.
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