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ARÁNZAZU MEDIERO Y COLS. AN. R. ACAD. NAC. FARM.

2.2. P2Y2 silencing

To design P2Y2 receptor-specific siRNA duplexes, rabbit P2Y2
receptor coding sequence (GenBank accession number EU886321)
was submitted to the Ambion siRNA target Finder website (http://
www.ambion.com/techlib/misc/siRNA_finder.html) for siRNA
prediction. One sequence of nine (max. GC content 60%) suggested
candidates was selected (6). Nucleotide sequence of the siRNA target
sites was as follows: P2Y2 siRNA #2, 5’-AACCTGTACTGCAGCATCCTC-
3’. This siRNAs was obtained from Applied Biosystems, in annealed
and lyophilized forms and were suspended in 0.9% NaCl before in
vivo use.

2.3. In vivo delivery of P2Y2 siRNA and wounding procedure

The siRNA was applied in one single eye in 10 nmol 0.9% NaCl
drops (volume instilled 40 µl) along four consecutive days. The
contralateral eye received the same volume of saline solution (0.9%
NaCl). Slit-lamp biomicroscopy was performed during instillation
process to evaluate possible changes in the cornea.

Corneal wounds were performed 10 hours before the fourth siR-
NA instillation. After topical anaesthesia (0.4% oxibuprocaine and 1%
tetracaine, Alcon Cusi, Barcelona, Spain), corneal wound were made
to the epithelium of both eyes by applying a 5-mm disc of Whatman
no. 1 paper soaked in n-heptanol (Sigma-Aldrich, St. Louis, MO) as
previously described (3). Briefly, discs were place in the centre of the
cornea and left there for 30 seconds (7) and after removal of the disc,
the eyes were washed with isotonic saline solution.

Ap4A treatment was performed every six hours as described
previously (3).

2.4. Immunohistochemistry

12, 24 and 36 hours after epithelium wounding (72, 84 and 96
hours after the first siRNA instillation), rabbits were euthanized with
sodium pentothal and eyes were enucleated. Corneas were dissected

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