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T*
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T*
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[(Ci)342.1(n5)342.1(%C)342.1(O)]TJ
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(2)Tj
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[(day)-335.7(8)-334.6(p.i.)]TJ
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[(FGF4)-341.2(grow)-7.4(th)-333.6(facto)-7.8(r)-332.8(supp)-6.4(lementat)-8.7(ion)]TJ
/F4 1 Tf
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[(To)-340.9(test)-343.6(the)-345.2(functional)-346.5(importance)-343.6(of)-344.7(miR-218,)-342.4(bovine)-343.5(IVP)]TJ
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[(embryos)-504.3(were)-495.3(grown)-499.8(in)-500.1(the)-496.9(presence)-498.9(of)-496.4(1)]TJ
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(m)Tj
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[(g/mL)-494.9(human)]TJ
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[(recombinant)-584(FGF4)-576.2(\(R&D)-582.9(Systems\))-579.3(and)-581.3(1)]TJ
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19.5315 0 TD
(m)Tj
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[(g/mL)-577(heparin)]TJ
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[(\(H3149;)-551.5(Sigma-Aldrich\))-550.4(as)-544.6(previously)-550.8(described)-552.5(by)-540(Kuijk)]TJ
T*
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[(culture)-449.2(medium)-445.5(was)-445.1(included)-447(for)-446.5(each)-444.7(of)-445.8(the)-446.4(3)-442.1(replicated)]TJ
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[(experiments.)-297.6(The)-297.1(cleavage)-291.3(rate)-291.4(was)-293.3(analyzed)-291(at)-290.8(48)-169.7(h)-287.9(p.i.)-295.2(and)]TJ
T*
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[(tocysts)-370.4(were)-368.9(selected)-367.7(for)-364.3(RT-qPCR)-365.6(analysis)-364.3(or)-363.9(for)-370.7(immuno-)]TJ
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[(uorescent)-340.9(staining)-335.7(as)-335.9(further)-335.8(described.)]TJ
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[(mi)-7.4(RNA)-339.2(expressi)-8.9(on)-335.7(analysis)]TJ
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[(Early)-403.2(blastocysts)-398.2(were)-406.8(selected)-399.3(at)-398.3(day)-399(7)-404.2(p.i)-399.8(and)-397.9(hatched)]TJ
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[(blastocysts)-341.3(were)-337.2(selected)-336.1(at)-328.8(day)-335.7(8)-334.6(p.i.)]TJ
1.1128 -1.2203 TD
[(Only)-558.2(embryos)-554.9(with)-559.1(good)-556.7(morphological)-562.6(characteristics)]TJ
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[(were)-280.3(selected,)-275.8(washed)-274.3(3)]TJ
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[(in)-272.5(PBS,)-277.6(and)-271.5(stored)-273.7(individually)-273.4(or)]TJ
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[(in)-240.9(pools)-245.7(of)-237.2(10)-239.3(embryos)-245(in)-234.5(2)]TJ
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(m)Tj
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[(L)-243.3(o)0(f)-237.2(the)-244(lysis)-241.7(buffer)-241.3([10%)-235.1(RNasin)]TJ
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[(Plus)-450.2(RNase)-453.9(inhibitor)-449.4(\(Promega\),)-452.7(5%)-443.7(dithiothreitol)-455.1(\(Prome-)]TJ
T*
[(ga\),)-361.4(and)-366.3(0.8%)-357.6(Igepal)-364.9(CA-630)-361(\(Sigma-Aldrich\))-367.5(in)-361(RNase-free)]TJ
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[(C)-329(until)-341.1(RNA)-334.4(extraction.)]TJ
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[(Whole)-272.1(miRNA)-271(proling)-264.9(by)-268.7(stem-loop)-275.6(RT-qPCR.)]TJ
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[(Total)-268.5(RNA,)]TJ
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[(including)-335.3(the)-338.9(small)-327.9(RNA)-334.4(fraction)-338.9(was)-331.2(isolated)-336.5(from)-330.3(6)-328.3(pools)]TJ
T*
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[(10)-542.8(hatched)-540.1(bovine)-539.6(IVP)-543.7(blastocysts)-543.6(\(day)-538.5(8)-537(p.i.\))-542.3(using)-538.8(the)]TJ
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[(miRNeasy)-250.7(Mini)-243.2(Kit)-247.1(\(Qiagen\))-242.4(according)-249.8(to)-243.5(the)-244(manufacturers)]TJ
T*
[(instructions.)-302.7(The)-290.8(quality)-295.5(of)-294.1(the)-294.6(RNA)-296.5(was)-293.3(evaluated)-292.4(with)-293.5(the)]TJ
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[(Experion)-402.5(electrophoresis)-406.5(system)-403.3(using)-399.7(the)-402.1(high-sense)-406.7(RNA)]TJ
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[(chips)-268.1(\(Bio-Rad\).)-267(The)-265.5(gel)-260.8(electrophoresis)-273.8(image)-264.2(showed)-264.9(sharp)]TJ
T*
[(18S)-560.2(and)-549.7(28S)-553.9(ribosomal)-559.9(bands)-555.4(indicative)-554.3(of)-559.7(good)-550.4(quality)]TJ
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[(RNA.)-362.6(Unfortunately,)-359.8(the)-357.8(RNA)-353.4(concentrations)-367.9(were)-356.2(too)-353.8(low)]TJ
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[(The)-360.4(isolated)-368.1(RNA)-359.7(was)-362.9(reverse)-363.6(transcribed)-364(with)-363.1(the)-364.2(miR-)]TJ
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[(NA)-741.2(reverse)-736.6(transcription)-741.2(kit)-735.6(\(Applied)-741.7(Biosystems,)-739.3(Life)]TJ
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[(Technologies\))-723.2(in)-715.1(combination)-716.2(with)-717.2(a)-714(human)-719.7(stem-loop)]TJ
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[(Megaplex)-547(miRNA)-544.2(primer)-547.5(pool)-541.6(\(Applied)-545.7(Biosystems,)-549.6(Life)]TJ
T*
[(Technologies\))-343.8(consisting)-339.6(of)-338.4(primers)-340.2(for)-332.7(366)-333.6(miRNAs)-343.1(and)-334.7(18)]TJ
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[(endogenous)-287.8(controls)-286.1(as)-285.4(previously)-285.2(described)-286.9([28].)-281.1(Following)]TJ
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[(the)-623.4(RT)-614.2(reaction,)-619.5(the)-617.1(cDNA)-615.4(was)-615.8(preamplied)-620.3(using)-614.7(the)]TJ
T*
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0 -1.2266 TD
[(both)-675(from)-665.4(Applied)-671.7(Biosystems,)-669.8(as)-671.1(previously)-670.9(described)]TJ
0 -1.2203 TD
[([28].)-445.5(Preamplication)-449.4(of)-445.9(the)-446.4(cDNA)-444.7(was)-438.7(necessary)-446(because)]TJ
T*
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0 -1.2266 TD
[(preimplantation)-552.1(embryos.)-545.1(The)-543.7(preamplication)-550.5(procedure)]TJ
0 -1.2203 TD
[(was)-476.7(previously)-481.2(shown)-480.4(to)-477.5(substantially)-475.7(increase)-481.7(the)-478(detec-)]TJ
T*
[(tion)-572.9(sensitivity)-572.7(with)-571.8(limited)-568(quantication)-574.7(bias)-565.2([28].)-571.9(The)]TJ
/F1 1 Tf
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(2)Tj
45.3795 0 TD
[(GOOSSENS)-332.4(ET)-338.7(AL.)]TJ
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[(qPCR)-459.1(amplication)-460.1(of)-452.2(366)-453.8(mature)-461.1(miRNAs)-457(using)-456.6(miRNA)]TJ
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[(TaqMan)-255.2(assays)-254(\(Applied)-254.8(Biosystems,)-252.5(Life)-253.5(Technologies\).)-258.2(The)]TJ
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[(qPCR)-339(mixture)-334.5(contained)-337.7(4)]TJ
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11.925 0 TD
(m)Tj
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[(L)-331.8(o)0(f)-338.4(Universal)-336.5(qPCR)-332.6(mastermix)]TJ
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[(\(Applied)-393.9(Biosystems,)-397.9(Life)-392.6(Technologies\),)-391(3)]TJ
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(m)Tj
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.5501 0 TD
[(L)-388.7(o)0(f)-395.3(a)-385.2(1/15)-393.6(di-)]TJ
-19.8287 -1.2266 TD
[(lution)-432.4(of)-433.2(miRNA)-430.3(TaqMan)-432.3(assay)-429.7(\(Applied)-431.9(Biosystems,)-435.8(Life)]TJ
0 -1.2203 TD
[(Technologies\),)-460.5(a)13.1(n)0(d)-430(1)]TJ
/F9 1 Tf
9.516 0 TD
(m)Tj
/F4 1 Tf
.5438 0 TD
[(L)-439.3(o)0(f)-426.9(d)0(i)18.8(l)0(u)20.4(t)12.2(e)12.8(d)-445.6(pr)23.2(e)12.8(a)0(m)21.9(p)0(l)20.4(i)0()18.8(e)12.8(d)-439.3(c)0(D)18.3(N)12.7(A.)-434.3(Al)26.7(l)]TJ
-10.0598 -1.2203 TD
[(re)18.4(a)13.1(c)0(t)19.2(i)0(o)19(n)15.6(s)-412.3(w)0(e)23.8(r)0(e)-392.6(r)0(u)16.9(n)-408.1(o)16.2(n)-408.1(a)-410.5(7)13.1(9)13.1(0)0(0)19.9(H)12.7(T)-407.7(q)0(P)19.5(C)12.5(R)-408.9(c)13.4(y)14.6(c)0(l)16.2(e)12.8(r)-405.4(\()12.2(Ap)16.2(pl)20.4(i)15.4(e)0(d)-394.9(B)0(i)18.8(o)0(-)]TJ
0 -1.2266 TD
[(sy)19.6(st)23.5(em)21.5(s,)-352(Li)18.8(f)12.2(e)-360.3(Te)22.4(ch)16.3(n)15.6(o)0(l)19(o)0(g)24.4(i)0(e)15.5(s)0(\))-349.5(u)0(n)20.5(d)0(e)22.4(r)-361.1(t)12.2(h)0(e)-351(f)12.2(ol)19(lo)19(wi)26.5(ng)-355.6(c)13.4(y)0(c)21.6(l)0(i)24.5(n)0(g)-355.6(c)13.4(on)19.1(-)]TJ
0 -1.2203 TD
[(di)18.8(t)12.2(i)0(o)19(n)0(s)20.6(:)-363.3(10)-144.4(mi)17.9(n)-363.8(a)13.1(t)-367.2(9)0(5)]TJ
/F7 1 Tf
8.9343 0 TD
()Tj
/F4 1 Tf
.3857 0 TD
[(C)-366.9(f)12.2(ol)19(l)15.4(o)0(w)20.9(e)0(d)-357(b)0(y)-356.6(4)13.1(0)-366.2(c)0(y)21.6(c)0(l)22.5(e)0(s)-355.3(o)0(f)-357.3(1)0(5)-144.5(s)-368(a)13.1(t)-367.2(95)]TJ
/F7 1 Tf
16.2562 0 TD
()Tj
/F4 1 Tf
.3857 0 TD
(C)Tj
-25.9619 -1.2203 TD
[(an)16.1(d)-382.4(1)-157.6(mi)24.2(n)-389.1(a)13.1(t)-392.5(6)13.1(0)]TJ
/F7 1 Tf
6.9678 0 TD
()Tj
/F4 1 Tf
.3857 0 TD
[(C)12.5(.)-388.6(Mi)23(RN)14.8(As)-375.8(wi)20.1(t)12.2(h)-389.1(a)-385.2(C)12.5(q-)20.4(va)18.7(l)15.4(u)0(e)]TJ
/F7 1 Tf
12.8798 0 TD
(<)Tj
/F4 1 Tf
.7082 0 TD
[(3)13.1(2)-391.5(we)23.8(re)-380(c)13.4(o)0(n)19.1(-)]TJ
-20.9415 -1.2203 TD
[(si)20.4(de)22.4(re)18.4(d)-319.1(e)0(x)16.9(p)0(r)23.2(e)12.7(ss)16.3(e)12.8(d)-319.1(an)16.1(d)-319.1(m)0(i)24.2(R)0(N)21.1(A)-323.9(e)12.8(x)0(p)15.5(r)0(e)24.7(s)0(s)22.7(i)0(o)19(n)-319.5(da)22.8(ta)-316.1(we)23.8(re)-310.4(no)19.1(rm)20.7(a)13.1(l)0(-)]TJ
0 -1.2266 TD
[(iz)22.2(ed)-344.3(us)22.6(in)18.4(g)-352.2(t)12.2(he)-344.7(g)14.6(l)0(o)19(b)14.6(al)-344.5(m)15.1(e)0(a)19.6(n)-351.1(as)-342.3(de)22.4(sc)18.4(ri)21.1(b)14.6(e)0(d)-344.3([)12.2(29)19.9(])12.2(.)-350.7(Ne)19.1(xt)22.7(,)-357(w)0(e)-336.6(h)0(a)16.1(v)0(e)]TJ
0 -1.2203 TD
[(us)16.3(e)12.7(d)-407.7(a)-410.5(p)0(r)23.2(e)0(v)18.3(i)0(o)19(u)0(s)22.6(l)0(y)-400(d)16(es)17.8(c)13.4(r)0(i)21.1(b)0(e)21(d)-414(m)15.1(e)12.8(th)15.1(od)-397.8(f)12.2(o)0(r)-401.8(s)0(t)23.5(a)13.1(nd)18.9(a)13.1(r)0(d)21.6(i)0(z)22.3(i)0(n)18.4(g)-409.1(ge)21(ne)]TJ
T*
[(ex)16.9(pr)23.2(e)12.7(s)0(s)16.3(i)15.4(on)-366.6(da)22.8(t)12.2(a)-378.9(of)-363.6(su)22.6(bs)19.6(t)12.2(a)0(n)22.4(t)0(i)21.3(a)0(l)22.2(l)0(y)-368.4(v)0(a)25(r)0(i)21.1(a)13.1(bl)17.3(e)-372.9(b)0(i)23.7(o)0(l)19(o)0(g)24.4(i)0(c)22.5(a)0(l)-363.4(r)0(e)18.4(p)0(l)20.4(i)0(c)22.5(a)13.1(te)18.6(s)]TJ
0 -1.2266 TD
[([3)19(0)13.1(])0(.)-269(B)16(y)-276.3(p)0(e)17.7(r)0(f)24.1(o)0(r)21.8(m)0(i)17.9(n)15.6(g)-276.3(a)-277.7(st)23.5(an)22.4(da)16.5(rd)21.6(i)15.4(z)0(a)19.9(t)12.2(io)19(n)-275.3(p)0(r)23.2(o)0(c)16.9(e)12.8(du)20.9(re)-266.2(b)14.6(a)13.1(se)17.8(d)-274.9(o)0(n)-265.4(l)0(o)19(g)]TJ
0 -1.2203 TD
[(tr)17.8(a)13.1(n)0(s)20.6(f)12.2(or)15.5(m)15.1(a)13.1(ti)21.3(on)19.1(,)-344.4(m)0(e)21.5(a)13.1(n)-344.8(c)0(e)19.8(n)0(t)21.4(e)12.8(ri)21.1(ng)17.5(,)-344.4(a)13.1(nd)-335.2(a)13.1(u)0(t)23.5(o)0(s)21.2(c)0(a)20.2(l)0(i)24.6(n)0(g)17.5(,)-344.4(i)15.4(n)0(t)15.1(e)12.7(re)24.7(xp)15.5(e)12.7(r)0(i)21.1(-)]TJ
T*
[(me)21.5(nt)21.4(al)-363.4(va)18.7(ri)21.1(a)13.1(t)12.2(io)19(n)-370.1(w)0(a)17.8(s)-368(ma)21.9(xi)19.6(m)15.1(a)0(l)22.2(l)0(y)-362(r)0(e)18.4(d)0(u)20.9(c)13.4(ed)22.4(.)-376(O)0(n)25.2(l)0(y)-362(m)0(i)17.9(R)14.7(NA)17.6(s)-368(e)0(x)16.9(-)]TJ
0 -1.2266 TD
[(pr)16.9(e)12.8(s)0(s)22.7(e)0(d)-262.1(i)0(n)-266.2(a)13.1(t)-272.4(l)0(e)21.9(a)0(s)18.1(t)-272.4(v)21.6(e)-271.8(o)0(u)21.2(t)-272.4(of)-262.5(th)21.4(e)-278.1(s)0(i)20.4(x)-267.7(te)18.6(st)23.5(ed)-262.1(sa)18.1(mp)20(l)15.4(e)0(s)-266.8(p)0(e)24(r)-272.6(gr)20.2(ou)21.2(p)]TJ
0 -1.2203 TD
[(we)17.4(re)-449.5(c)13.4(o)0(n)19.1(s)0(i)20.4(d)0(e)22.4(r)0(e)18.4(d)-458.2(f)12.2(o)0(r)-452.4(d)16(ow)20.9(ns)20.6(tr)17.8(e)12.8(a)13.1(m)-465.5(a)13.1(na)22.4(ly)23.7(si)20.4(s.)-446.8(Mi)16.6(R)14.7(N)0(A)17.6(s)-456.6(wi)20.1(th)-452.8(a)]TJ
T*
[(fo)15.7(l)15.4(d)-363.4(di)25.1(ff)18(e)12.7(r)0(e)18.4(n)0(c)22.6(e)]TJ
/F7 1 Tf
6.8414 0 TD
(>)Tj
/F4 1 Tf
.7082 0 TD
[(2)-359.9(a)13.1(nd)-354.1(a)]TJ
/F5 1 Tf
3.7368 0 TD
(P)Tj
/F4 1 Tf
.9611 0 TD
[(va)18.7(lu)20.4(e)]TJ
/F7 1 Tf
2.8896 0 TD
()Tj
/F4 1 Tf
.7082 0 TD
[(0)13.1(.)0(0)16.5(5)-359.9(\()12.2(t)12.2(h)0(e)-351(M)0(a)20.7(n)0(n)18.5()13.1(Wh)22.9(it)21.3(ne)15.7(y)]TJ
-15.8452 -1.2203 TD
[(te)18.6(st)23.5(\))-354.6(w)0(e)23.8(r)0(e)-342(c)13.4(on)19.1(si)20.4(d)16(e)0(r)18.4(e)12.8(d)-350.8(a)13.1(s)-349.1(d)0(i)18.8(f)12.2(fe)18.6(re)24.7(nt)15.1(i)15.4(a)0(l)22.2(l)0(y)-343.1(e)12.8(xp)21.8(re)18.4(ss)22.7(e)12.8(d)-350.8(b)14.6(e)0(t)18.6(w)0(e)23.8(e)0(n)-344.7(t)12.2(he)]TJ
0 -1.2266 TD
[(ea)19.6(rl)21.1(y)-320.6(a)13.1(nd)-316.2(t)12.2(h)0(e)-313.1(h)0(a)22.4(t)12.2(ch)16.3(e)12.8(d)-319.1(bl)17.3(a)13.1(s)0(t)23.5(o)0(c)16.9(y)14.6(st)-311.6(gr)20.2(ou)21.2(p.)]TJ
1.1128 -1.2203 TD
[(Hierarchical)-607.2(cluster)-600.9(analysis)-604.6(was)-603.1(performed)-600.7(using)-602(the)]TJ
-1.1128 -1.2203 TD
[(Multi)-336.5(Experiment)-340.8(Viewer)-330.9(\()-89(MEV)-333.4(4.6.2\))-333.6(software)-335.6([31].)]TJ
/F5 1 Tf
1.1128 -1.2266 TD
[(In)20.4(di)17.6(vi)17.6(d)13.1(u)0(a)15.3(l)-438.2(mi)22(RN)13.3(A)-439.1(d)13.1(et)17.8(ec)19.6(ti)16.6(o)13.4(n)-440.7(by)-433(S)14.6(Y)0(B)18.1(R)-440.5(gr)18.8(ee)17.6(n)14.6(-)0(b)14.9(a)13.4(se)17.6(d)-442.1(R)14.7(T-)15.5(qP)18.7(CR)23.2(.)]TJ
/F4 1 Tf
-1.1128 -1.2203 TD
[(RNA,)-400.6(isolated)-399.7(as)-399.2(described)-400.7(before,)-403.1(was)-400.8(reverse)-401.5(transcribed)]TJ
T*
[(using)-488.2(the)-484.3(miRCURY)-489.6(locked)-487.1(nucleic)-484.5(acid)-485.9(\(LNA\))-485.9(Universal)]TJ
0 -1.2266 TD
[(cDNA)-248.7(synthesis)-246.7(kit)-248.7(\(Exiqon\).)-244.4(Mature)-247.3(miRNA)-240.7(expression)-250.3(was)]TJ
0 -1.2203 TD
[(quantied)-410.6(using)-412.4(the)-408.4(SYBR)-408.3(Green)-407.3(master)-410.5(mix)-407.9(\(Exiqon\))-412.2(and)]TJ
T*
[(specic)-425(LNA)-428(PCR)-423.1(primer)-421.1(sets)-426.6(for)-427.6(miR-218,)-424.6(miR-449b,)-422.7(and)]TJ
0 -1.2266 TD
[(miR-155)-491.2(\(Exiqon\).)-491(U6)-487.8(snRNA,)-487.5(5SrRNA,)-494.1(and)-486.4(RNU1A)-493.4(\(Ex-)]TJ
0 -1.2203 TD
[(iqon\))-419.2(were)-413.1(chosen)-414.7(as)-411.8(reference)-415.1(small)-416.4(RNAs)-411.6(for)-414.9(normaliza-)]TJ
T*
[(tion,)-335.5(as)-335.9(described)-337.5(in)-329.4([32].)]TJ
/F8 1 Tf
9.9626 0 0 9.9626 59.7543 339.2503 Tm
[(Targ)-9(et)-333.6(gene)-335.7(pred)-8.4(iction,)-340(pathwa)-8.1(y)-330.8(analysi)-9.3(s,)]TJ
0 -1.2007 TD
[(and)-341.4(expressio)-8.9(n)-330(anal)-9.6(ysis)]TJ
/F4 1 Tf
8.9663 0 0 8.9663 69.7323 309.4298 Tm
[(Ca)19.3(nd)18.9(id)18.8(a)13.1(t)0(e)-253.3(t)0(a)19(r)0(g)20.2(e)0(t)-253.3(g)0(e)14.7(n)0(e)22(s)-266.9(we)23.8(re)-253.5(pr)16.9(e)12.8(d)0(i)18.8(c)0(t)19.2(e)0(d)-255.8(f)12.2(or)-256.4(t)12.2(h)0(e)-256.2(d)0(i)18.8(f)12.2(fe)18.6(re)18.4(nt)21.4(ia)15.9(ll)18.2(y)]TJ
-1.1128 -1.2203 TD
[(ex)16.9(pr)16.9(e)12.8(s)0(s)16.3(e)12.8(d)-572.1(m)15.1(iR)17.5(NA)17.6(s)-570.4(u)0(s)22.6(i)0(n)18.4(g)-567.2(a)-568.6(c)13.4(om)18.6(bi)17.3(na)22.4(ti)15(on)-556.2(of)-559.7(t)12.2(h)0(r)14.9(e)12.7(e)-569(m)0(i)17.9(R)14.7(NA)]TJ
0 -1.2266 TD
[(ta)19(rg)20.2(et)-392.4(g)14.6(e)0(n)15.7(e)-391.9(pr)16.9(ed)16.1(i)15.4(c)0(t)12.9(i)15.4(on)-391.9(da)22.8(ta)19(ba)21.4(se)17.8(s)-393.3(\()0(T)15.5(a)13.1(rg)20.2(et)18.6(Sc)14.9(a)13.1(n)0(,)-385.7(m)0(i)17.9(R)0(a)21.6(n)0(d)18.9(a)0(,)-388.1(a)13.1(nd)]TJ
0 -1.2203 TD
[(Pi)14.1(c)13.4(T)0(a)16.5(r)0(-)24.1(4)0(w)17.8(a)0(y)21.4(\))-436.8(on)-429.8(t)12.2(h)0(e)-426.9(G)0(O)17.2(m)15.1(ir)-427.9(we)17.4(b)14.6(s)0(i)14.1(t)12.2(e)-436.2(\()0(w)16.9(w)0(w)22(.)0(b)18(i)0(o)19(a)13.1(ca)20.2(de)16.1(my)23.3(.g)18(r/)]TJ
T*
[(bi)17.3(oi)19(nf)21.4(or)15.5(ma)21.9(ti)15(c)13.4(s)0(/)21(p)0(r)16.9(o)0(j)17.6(e)12.7(ct)19.2(s/)14.7(G)13.9(O)0(m)18.4(i)0(r)21.1(\))0(.)-287.9(N)12.7(ex)16.9(t)12.2(,)-293.8(a)-296.7(s)0(e)24.1(l)0(e)15.5(c)13.4(ti)15(on)-284.4(wa)24.2(s)-298.5(m)15.1(ad)16.5(e)12.8(,)]TJ
T*
[(ba)15(se)24.1(d)-433(o)0(n)-417.1(t)12.2(he)-420.6(re)18.4(su)16.3(lt)21.3(s)-431.3(o)0(f)-420.6(t)12.2(he)-420.6(in)18.4(t)12.2(e)0(r)18.4(s)0(e)17.8(c)0(t)19.2(i)0(o)19(n)0(s)-422(b)14.6(et)18.6(we)17.4(en)-420.6(t)12.2(h)0(e)-420.6(t)0(a)19(r)0(g)20.2(e)0(t)]TJ
0 -1.2266 TD
[(ge)14.7(ne)-509.1(pr)23.2(ed)16.1(ic)22.5(ti)15(on)-505.7(pr)16.9(og)18.1(ra)25.1(ms)-511(an)16.1(d)-515.2(o)0(n)-505.7(t)0(h)15.1(e)-518.4(e)12.8(x)0(p)15.5(e)12.8(ri)14.7(m)15.1(e)0(n)15.7(t)12.2(al)15.9(ly)-507.5(su)16.3(p-)]TJ
0 -1.2203 TD
[(po)14.8(rt)17.8(e)12.8(d)-236.9(m)15.1(i)0(R)17.5(N)0(A)17.6(-)12.2(mR)17.2(NA)-222.7(in)18.4(te)18.6(ra)18.8(ct)19.2(io)19(ns)-226(a)13.1(s)-235.3(re)18.4(po)14.8(rt)24.1(ed)-224.2(by)-223.8(Ta)16.5(rb)20.2(a)13.1(s)0(e)17.8(.)-236.9(At)]TJ
T*
[(th)15.1(e)-480.4(s)0(a)18.1(m)15.1(e)-480.4(t)0(i)15(m)15.1(e,)-470.7(sp)16.3(e)12.8(c)0(i)16.2()0(c)-470.2(g)14.6(en)15.7(e)-480.4(f)12.2(un)14.2(c)13.4(t)0(i)15(o)0(n)19.1(s)-475.5(we)17.4(re)-468.5(ta)19(ke)14.7(n)-477.6(i)0(n)18.4(t)12.2(o)-483.3(a)13.1(c-)]TJ
0 -1.2267 TD
[(co)16.9(un)20.6(t,)-319.5(ba)21.4(se)17.8(d)-325.5(o)0(n)-322.3(I)0(n)19.8(g)0(e)21(n)0(u)20.6(i)0(t)15(y)-326.9(Pa)18.1(t)12.2(h)0(w)20.3(a)0(y)-320.1(A)0(n)20.5(a)0(l)15.9(y)14.6(si)20.4(s)-330.1(\()0(I)16.4(P)0(A)22.5(;)-325.4(In)13.5(g)14.6(e)0(n)15.7(u)0(i)20.4(t)0(y)]TJ
0 -1.2203 TD
[(Sy)16.1(st)17.2(e)12.8(m)0(s)20.1(,)-445.5(ww)15.7(w.)20.7(in)18.4(g)14.6(e)0(n)15.7(u)0(i)20.4(t)0(y)20.4(.)0(c)16.8(o)0(m)18.6(\))-443.1(an)16.1(d)-445.6(o)0(n)-436.1(l)0(i)18.2(t)12.2(er)18.4(at)19(ur)16.9(e)12.7(.)-445.5(Us)16.3(in)18.4(g)-447(t)12.2(hi)18.4(s)]TJ
T*
[(st)17.2(ra)18.8(t)12.2(e)0(g)14.7(y)14.6(,)-268.5(1)0(8)19.9(7)-271.4(po)21.2(t)12.2(e)0(n)15.7(t)12.2(ia)15.9(ll)18.2(y)-263.7(i)0(n)18.4(t)0(e)18.6(r)0(e)18.4(s)0(t)17.2(i)0(n)18.4(g)-263.7(ta)19(rg)20.2(et)-259.6(ge)21(ne)15.7(s)-266.9(w)0(e)17.4(r)0(e)-253.5(s)0(e)17.8(l)0(e)15.5(c)13.4(te)18.6(d)]TJ
0 -1.2266 TD
[(fo)15.7(r)-323.2(g)0(e)21(n)0(e)-313.1(e)0(x)16.9(p)0(r)16.9(e)12.7(ss)16.3(io)19(n)-325.9(a)13.1(na)16.1(l)15.4(y)0(s)19.6(i)0(s)-314.7(b)0(y)-312.3(R)0(T)18.1(-)0(q)20.4(P)0(C)17.4(R)0(.)]TJ
1.1128 -1.2203 TD
[(For)-463.1(the)-465.3(target)-460.7(gene)-469.2(expression)-465.3(analysis,)-462(total)-468.3(RNA)-460.9(was)]TJ
-1.1128 -1.2203 TD
[(isolated)-292.2(from)-279.7(8)-284(pools)-290(of)-281.4(10)-283.6(early)-286.3(IVP)-284.5(bovine)-286.6(blastocysts)-290.7(\(day)]TJ
T*
[(7)-296.7(p.i.\))-289.4(and)-296.8(8)-290.4(pools)-296.3(of)-294.1(10)-289.9(hatched)-293.5(bovine)-299.3(IVP)-290.8(blastocysts)-297(\(day)]TJ
0 -1.2266 TD
[(8)-303(p.i.\))-302(using)-304.9(the)-307.3(RNeasy)-300.2(Micro)-302.3(Kit)-304(\(Qiagen\))-305.6(according)-306.7(to)-300.4(the)]TJ
0 -1.2203 TD
[(manufacturers)-285.6(protocol.)-275.8(After)-281.1(RNA)-271.2(extraction,)-280.7(an)-274.8(RT)-279.1(minus)]TJ
T*
[(control)-278.5(PCR)-277.6(was)-274.3(performed)-278.2(with)-274.6(primers)-277(for)]TJ
/F5 1 Tf
20.031 0 TD
(GAPDH)Tj
/F4 1 Tf
3.857 0 TD
[([33])-271.9(to)]TJ
-23.888 -1.2266 TD
[(check)-486.8(the)-490.6(RNA)-479.8(for)-490.8(genomic)-485.1(DNA)-483.3(contamination)-487.5(and)-486.4(the)]TJ
0 -1.2203 TD
[(RNA)-492.5(quantity/quality)-486.7(assessment)-492.9(was)-489.3(done)-483.3(with)-489.6(the)-490.6(Ex-)]TJ
T*
[(perion)-277.2(\(Bio-Rad\).)-279.6(The)-278.2(rst-strand)-279.9(cDNA)-273.9(synthesis)-284.6(and)-271.5(linear)]TJ
0 -1.2267 TD
[(amplication)-441.1(were)-432.1(done)-439(using)-437.6(the)-433.7(WT-Ovation)-439.6(RNA)-435.6(Am-)]TJ
0 -1.2203 TD
[(plication)-500.9(system)-498.2(\(NuGEN\))-503.5(as)-494(described)-501.9(in)-493.8(the)-503.3(manufac-)]TJ
28.0042 74.5471 TD
[(turers)-562.7(instructions.)-568.2(After)-565.7(the)-560.2(RT)-563.7(reaction)-559.7(and)-562.3(the)-566.5(linear)]TJ
0 -1.2203 TD
[(amplication)-333.6(step,)-334.8(the)-338.9(cDNA)-337.2(was)-331.2(diluted)-337.3(20)-334.1(times.)]TJ
1.1128 -1.2266 TD
[(The)-335.1(qPCR)-339(reactions)-339.7(consisted)-337.4(of)-338.4(1)]TJ
/F9 1 Tf
15.3647 0 TD
(m)Tj
/F4 1 Tf
.5501 0 TD
.3318 Tc
(L5)Tj
/F7 1 Tf
1.606 0 TD
0 Tc
()Tj
/F4 1 Tf
.7208 0 TD
[(Real-Time)-334.1(ready)]TJ
-19.3544 -1.2203 TD
[(DNA)-369.5(Probes)-370.9(Master)-367.4(\(Roche\),)-373.8(0.25)]TJ
/F9 1 Tf
15.3204 0 TD
(m)Tj
/F4 1 Tf
.5564 0 TD
[(L)-363.4(LightCycler)-376.9(480)-365.3(Reso-)]TJ
-15.8768 -1.2203 TD
[(Light)-406.8(Dye)-416.7(\(Roche\),)-411.8(1)]TJ
/F9 1 Tf
9.5982 0 TD
(m)Tj
/F4 1 Tf
.5564 0 TD
[(L)-407.7(forward)-413.5(primer)-408.4(\(1.25)]TJ
/F9 1 Tf
10.6288 0 TD
(m)Tj
/F4 1 Tf
.5564 0 TD
[(M\),)-406.8(1)]TJ
/F9 1 Tf
2.5861 0 TD
(m)Tj
/F4 1 Tf
.5501 0 TD
[(L)-407.7(re-)]TJ
-24.476 -1.2266 TD
[(verse)-413.5(primer)-414.7(\(1.25)]TJ
/F9 1 Tf
8.3526 0 TD
(m)Tj
/F4 1 Tf
.5501 0 TD
[(M;)-412.7(IDT\),)-414.6(and)-410.6(1.75)]TJ
/F9 1 Tf
8.302 0 TD
(m)Tj
/F4 1 Tf
.5501 0 TD
[(L)-414(o)0(f)-407.9(diluted)-419.5(cDNA.)]TJ
-17.7547 -1.2203 TD
[(Primers)-295.9(used)-302.7(for)-294.8(qPCR)-301(are)-297.3(listed)-296.3(in)-297.8(Supplementary)-302.8(Table)-301.2(S1)]TJ
T*
[(\(Supplementary)-771.2(Data)-766(are)-771.5(available)-766.6(online)-768.9(at)-765(www.lie-)]TJ
0 -1.2266 TD
[(bertpub.com/scd\).)-423.5(All)-419.5(assays)-418.4(were)-419.4(run)-416.4(on)-417.1(a)-416.8(CFX384)-418.3(Real-)]TJ
0 -1.2203 TD
[(Time)-332.8(PCR)-334.5(detection)-335.2(system)-340.1(\(Bio-Rad\).)]TJ
1.1128 -1.2203 TD
[(O)15.9(n)0(l)18.4(y)-333.2(a)13.1(s)0(s)16.3(a)13.1(ys)-321.9(wi)20.1(th)-320(go)18.1(od)-321.9(PC)23.8(R)-333(e)12.7(f)15.5(ci)22.5(en)15.7(cy)-319.8(\()]TJ
/F7 1 Tf
17.565 0 TD
(>)Tj
/F4 1 Tf
.7082 0 TD
-.0131 Tc
[(80)-13.1(%)]TJ
/F7 1 Tf
1.9664 0 TD
0 Tc
(<)Tj
/F4 1 Tf
.7082 0 TD
[(12)19.9(0%)17.8(\))-329.3(a)0(n)16.1(d)]TJ
-22.0606 -1.2203 TD
[(st)17.2(a)13.1(n)0(d)18.9(a)13.1(rd)-560.1(de)22.4(vi)21(at)19(io)19(ns)-554.8(be)14.7(l)15.4(o)0(w)-554.5(0)0(.)16.5(5)-562.3(be)21(tw)16.9(ee)19.2(n)-566.1(s)0(a)18.1(m)15.1(pl)14.1(e)-562.6(d)0(u)20.9(p)0(l)20.4(i)0(c)16.2(a)13.1(te)18.6(s)]TJ
0 -1.2266 TD
[(we)17.4(re)-455.8(t)12.2(a)0(k)21.4(e)0(n)-458.5(i)0(n)18.4(t)12.2(o)-470.7(a)13.1(cc)20.4(ou)14.8(nt)-452.8(\(S)13.7(up)22.6(pl)20.4(em)15.2(e)12.8(n)0(t)15.1(a)13.1(ry)-460.4(Ta)22.8(bl)17.3(e)-461.5(S)0(1)14.7(\))12.2(.)-464.5(A)0(s)16.3(s)0(a)18.1(y)14.5(s)]TJ
0 -1.2203 TD
[(t)12.2(h)0(a)16.1(t)-259.7(di)18.8(d)-262.2(n)0(o)12.8(t)-259.7(me)15.2(e)12.7(t)-266(t)12.2(h)0(e)15.7(s)0(e)-254.1(c)0(r)19(i)0(t)21.3(e)0(r)18.4(i)0(a)-256(w)0(e)17.4(r)0(e)-253.5(e)12.7(xc)17.5(lu)20.4(de)16.1(d)-262.2(f)12.2(ro)15.5(m)-263.1(t)12.2(he)-256.2(st)17.2(ud)20.9(y.)]TJ
1.1128 -1.2203 TD
[(Combined)-596.5(a)32.1(n)28.2(a)25.8(l)28.1(y)27.2(s)24(i)28.1(s)-564(o)28.8(f)-569.5(m)27.7(i)28.1(R)27.4(N)25.3(A)-564.1(a)25.8(n)28.2(d)-565.7(m)27.7(R)27.4(N)25.3(A)-570.5(p)30.3(r)24.6(o)28.9()28.6(l)28.1(e)25.4(s)-564(w)23.7(a)32.1(s)]TJ
-1.1128 -1.2266 TD
-.0276 Tc
[(carried)-428.9(o)1.2(ut)-432.7(b)5.9(y)-430.4(in)-5.7(vestigatin)-5.7(g)-424(t)-2.8(he)]TJ
14.0432 0 TD
-.0265 Tc
[(coexpr)4.4(ession)-428.2(of)-431.6(the)-431.1(p)3.8(re)5.2(dic)5.8(t)-1.7(ed)]TJ
-14.0432 -1.2203 TD
-.0282 Tc
[(miRNA-mRNA)-377.4(pairs)-377.3([)-3.4(34])-376.4(w)-4.5(here)]TJ
13.8851 0 TD
[(by)-374(those)-375.8(t)-3.3(arget)-376.3(g)-.9(en)-6.2(es)-377.2(with)-379.2(an)]TJ
-13.8851 -1.2203 TD
-.0258 Tc
[(inve)5.9(rs)4.5(e)-354.5(e)5.9(xp)4.5(re)5.9(ss)4.5(ion)-351.7(p)4.5(at)5.3(ter)5.1(n)-351.7(compa)6.3(r)-1.2(ed)-344.9(to)-351(the)5.9(i)-4(r)-349(t)5.3(arg)7.7(e)-.4(ting)-352.7(miRN)5.8(A)]TJ
0 -1.2266 TD
-.0278 Tc
[(were)-261.6(selected)-252.1(for)-256.1(f)-3(urther)-256.1(validatio)]TJ
13.961 0 TD
-.0302 Tc
[(n)-254.9(b)-3(y)-255.9(l)-8.4(uc)-4.2(if)-5.4(er)-5.6(a)-4.4(s).1(e)-257.7(r)-5.6(ep)-6.3(or)-5.6(te)-4.8(r)-258.5(a)-4.4(ss)-6.2(ays)-6.2(.)]TJ
/F8 1 Tf
9.9626 0 0 9.9626 310.8472 502.6392 Tm
0 Tc
[(Luciferase)-341.6(report)-8.9(er)-332.8(assa)-7.2(y)]TJ
/F5 1 Tf
8.9663 0 0 8.9663 320.8251 484.781 Tm
[(Wild-type)-281(3)]TJ
/F7 1 Tf
4.7864 0 TD
()Tj
/F5 1 Tf
.2529 0 TD
[(UTR)-280.5(constructs.)]TJ
/F4 1 Tf
7.7266 0 TD
[(The)-278.2(hypothesized)-288.5(interaction)]TJ
-13.8788 -1.2203 TD
[(between)-306.8(a)-309.3(miRNA)-303.9(and)-309.4(its)-308.8(candidate)-308.8(target)-309(was)-306(tested)-308.5(using)]TJ
0 -1.2266 TD
[(an)-394.9(in)-392.6(vitro)-393.9(luciferase)-401.5(assay)-391.8(as)-399.2(previously)-392.7(described)-400.7([35,36].)]TJ
0 -1.2203 TD
[(The)-284.5(3)]TJ
/F7 1 Tf
2.428 0 TD
()Tj
/F4 1 Tf
.2466 0 TD
[(UTR)-286.8(segments)-286.5(of)-287.8(the)-282(target)-283.7(genes)-287.1(were)-286.7(PCR)-284(amplied)]TJ
-2.6746 -1.2203 TD
[(using)-273.2(bovine)-267.7(blastocyst)-276.7(cDNA)-273.9(as)-272.7(a)-265.1(template)-274.6(and)-271.5(the)-275.6(primers)]TJ
0 -1.2266 TD
[(listed)-252(in)-247.2(Table)-250.6(1.)-249(The)-246.6(primers)-251.7(are)-253(anked)-247.2(by)-249.1(either)-254.5(an)]TJ
/F5 1 Tf
23.5023 0 TD
(Xho)Tj
/F4 1 Tf
1.6566 0 TD
.2487 Tc
[(Io)248.7(r)]TJ
-25.1589 -1.2203 TD
0 Tc
(a)Tj
/F5 1 Tf
.9611 0 TD
(Not)Tj
/F4 1 Tf
1.5301 0 TD
[(I)-463.7(restriction)-465(site)-466.8(at)-461.6(their)-463.2(5)]TJ
/F7 1 Tf
11.8997 0 TD
()Tj
/F4 1 Tf
.7082 0 TD
[(to)-464.8(allow)-466.2(ligation)-463.8(into)-459.1(the)]TJ
-15.0991 -1.2203 TD
[(multiple)-378(cloning)-380.7(region)-381.4(of)-376.3(the)-383.1(psiCHECK-2)-379.1(Vector)-379(\(Prome-)]TJ
T*
[(ga\))-484.9(downstream)-484.5(of)-490.1(the)-484.3(stop)-486.5(codon)-485.4(of)-490.1(an)-483.4(SV40)-487.2(promoter-)]TJ
0 -1.2266 TD
(driven)Tj
/F5 1 Tf
3.2057 0 TD
(Renilla)Tj
/F4 1 Tf
3.1867 0 TD
[(luciferase)-338.3(gene.)]TJ
-5.2796 -1.2203 TD
[(H)12.7(E)0(K)19.5(2)0(9)19.9(3)0(T)-558.9(c)0(e)19.8(l)0(l)18.2(s)-570.4(we)17.4(re)-557(se)17.8(ed)16.1(e)12.7(d)-572.1(in)-557(th)15.1(e)-569(R)0(P)19.7(M)0(I)-563.6(m)15.1(ed)16.1(iu)20.4(m)-572.9(\()12.2(In)19.8(-)]TJ
-1.1128 -1.2203 TD
[(vi)21(tr)17.8(og)18.1(e)12.8(n)0(,)-575.4(L)0(i)18.8(f)0(e)-582.1(T)0(e)22.4(c)0(h)16.3(n)0(o)19.1(l)0(o)19(g)0(i)23.7(e)0(s)17.8(\))-588.5(su)16.3(pp)22.6(le)15.5(me)21.5(nt)15.1(e)12.8(d)-584.7(wi)20.1(th)-579.2(10)19.9(%)-589.7(F)14.6(CS)]TJ
0 -1.2266 TD
[(\()12.2(I)0(n)19.8(v)0(i)14.7(t)12.2(ro)15.5(g)14.5(e)0(n)15.7(,)-445.5(Li)18.8(f)12.2(e)-448.8(T)16(e)0(c)13.5(h)0(n)18.5(o)0(l)19(o)0(g)24.4(i)0(e)15.5(s)0(\))-438.1(a)13.1(t)-443.1(a)-448.4(de)22.4(ns)14.3(it)21.3(y)-447(o)0(f)-433.2(1)0(0)19.9(,)0(0)16.5(0)13.1(0)-448.4(c)13.4(el)15.5(l)15.4(s)0(/)]TJ
0 -1.2203 TD
[(we)17.4(l)15.4(l)-503(in)-487.5(a)13.1(n)-502.9(op)21.2(aq)21.4(ue)-494.4(9)13.1(6)0(-)19(w)0(e)17.4(l)0(l)-487.6(p)0(l)20.4(a)0(t)19(e)-499.4(\()12.2(N)0(u)17.6(n)0(c)16.3()13.1(Th)18.9(er)18.4(mo)-493.5(S)14.2(c)0(i)16.2(e)12.7(nt)15.1(i)18.8(c)13.4(\))0(.)]TJ
T*
[(Tw)20.7(e)12.7(n)0(t)15.1(y)0(-)20.4(f)0(o)15.7(u)0(r)-248.6(h)0(o)12.8(u)0(r)23.2(s)-266.9(a)13.1(f)0(t)18(e)0(r)-253.5(s)0(e)17.8(e)12.7(di)18.8(ng)17.5(,)-262.2(c)0(e)19.8(l)0(l)18.2(s)-260.5(we)17.4(re)-253.5(tr)17.8(an)16.1(sf)23.5(ec)19.8(te)12.3(d)-262.2(w)0(i)20.1(t)12.2(h)-262.6(a)]TJ
0 -1.2266 TD
(3)Tj
/F7 1 Tf
.4869 0 TD
()Tj
/F4 1 Tf
.2403 0 TD
[(UT)20.9(R)-333(c)0(o)16.9(n)0(t)21.4(a)0(i)15.9(n)0(i)24.7(n)0(g)-323.9(p)0(s)22.6(i)0(C)15.3(H)12.7(EC)15.8(K-)22(2)-328.3(V)0(e)16.3(c)0(t)19.2(o)0(r)-319.6(c)0(o)16.9(n)0(s)20.6(t)0(r)17.8(u)0(c)18.3(t)-329.3(\(3)19(45)-144.4(ng)17.5(\))-329.3(a)0(n)16.1(d)]TJ
-.7271 -1.2203 TD
[(t)12.2(h)0(e)-325.8(a)0(p)18.1(p)0(r)23.2(o)0(p)14.8(r)0(i)21.1(a)0(t)19(e)-335(mi)17.9(R)14.7(N)0(A)-323.8(p)0(r)16.9(e)0(c)19.8(u)0(r)16.9(s)0(o)21.2(r)-335.8(\()12.2(5)0(0)-150.8(n)0(M)23.1(,)-338(pr)23.2(e-)18.6(mi)17.9(R;)-323.3(Am)20(bi)17.3(on)19.1(,)]TJ
T*
[(Li)18.8(f)12.2(e)-569(te)18.6(ch)16.3(no)19.1(lo)19(gi)17.3(e)12.7(s)0(;)-560.6(h)0(s)20.6(a)0(-)19(p)0(r)16.9(e)12.8(-m)14.6(iR)23.8(-2)19(18)20(:)-572(P)0(M)18.8(1)13.1(03)19.9(28)19.9(;)-572(h)0(s)20.6(a)0(-)19(p)0(r)16.9(e)12.7(-m)20.9(iR)17.5(-)]TJ
0 -1.2266 TD
[(4)13.1(4)0(9)19.9(b)0(:)-627(P)0(M)18.8(1)0(1)19.9(5)0(2)20(1)0(;)-628.4(h)0(s)20.6(a)0(-)19(p)0(r)16.9(e)12.7(-m)14.6(iR)23.9(-1)19(30)20(a:)-628.4(PM)18.8(10)19.9(5)13.1(0)0(6)19.9(;)-641.5(hs)20.6(a-)19(pr)16.9(e)12.7(-)0(m)20.9(i)0(R)17.5(-)]TJ
0 -1.2203 TD
[(2)13.1(0)0(3)19.9(:)-376(PM)18.8(10)19.9(15)19.9(2\))-366.7(us)16.3(in)18.4(g)-377.5(0)13.1(.4)]TJ
/F9 1 Tf
10.8944 0 TD
(m)Tj
/F4 1 Tf
.5438 0 TD
[(L)-376(D)0(h)20.5(a)0(r)18.8(m)0(a)21.9(F)0(E)17.9(C)12.5(T)-376.1(D)0(u)16.2(o)-375.8(\(D)17.1(ha)22.4(rm)14.4(a)13.1(c)0(o)16.9(n)0(,)]TJ
-11.4381 -1.2203 TD
[(Th)18.9(e)12.7(r)0(m)20.7(o)-337.9(Sc)21.2(ie)15.5(nt)21.4(i)18.8(c\))19.2(.)-338(A)0(l)20.4(l)-332.3(tr)17.8(a)13.1(n)0(s)14.3(f)12.2(ec)19.8(ti)15(on)19.1(s)-336.4(w)0(e)23.8(r)0(e)-329.4(c)13.4(on)19.1(du)14.6(c)13.4(t)0(e)18.6(d)-338.1(t)12.2(w)0(i)20.1(c)0(e)-328(i)0(n)]TJ
T*
[(t)12.2(r)0(i)14.7(p)0(l)20.4(i)0(c)22.5(a)0(t)19(e)-448.8(wi)20.1(th)-440.1(th)15.1(e)-448.8(i)0(n)18.4(c)13.4(lu)14.1(si)20.4(on)-442.4(of)-439.5(a)-448.4(p)0(o)21.2(s)0(i)20.4(t)0(i)15(v)0(e)-436.9(\()0(v)17.8(e)12.7(ct)12.9(or)-433.4(on)12.8(l)15.4(y)0(\))-441.2(a)0(n)16.1(d)]TJ
0 -1.2266 TD
[(ne)22(ga)15(t)12.2(i)0(v)21(e)-505.7(co)16.9(nt)21.4(ro)15.5(l)-503(\()0(5)19(0)-157.6(nM)16.8(,)-502.4(A)0(m)20(b)0(i)17.3(o)0(n)-493(p)0(r)16.9(e)12.7(-m)14.6(iR)-494.6(ne)22(ga)15(t)12.2(i)0(v)21(e)-505.7(co)16.9(nt)21.4(ro)15.5(l)]TJ
0 -1.2203 TD
[(AM)18.8(1)13.1(7)0(1)19.9(1)0(1)19.9(,)-432.9(Am)20(b)14.6(i)0(o)19(n)-433.3(Li)18.8(f)12.2(e)-429.9(te)18.6(ch)16.3(no)19.1(lo)19(gi)17.3(e)12.8(s)0(\))17.2(.)-426.5(Ce)18.9(ll)18.2(s)-431.3(w)0(e)17.4(r)0(e)-417.9(l)0(y)17.3(s)0(e)17.8(d)-426.6(an)16.1(d)]TJ
T*
[(lu)20.4(mi)17.9(ne)22(sc)18.4(en)15.7(c)13.4(e)-335(wa)24.2(s)-336.4(q)14.5(ua)18.1(nt)15.1(i)18.8(e)12.8(d)-331.8(48)-144.5(h)-332.2(a)0(f)19(t)0(e)18.6(r)-335.8(t)12.2(r)0(a)18.8(n)0(s)20.6(f)0(e)18.6(c)0(t)19.2(i)0(o)19(n)-332.2(us)16.3(in)18.4(g)-333.2(t)12.2(he)]TJ
0 -1.2267 TD
[(Du)22.5(al)15.9(-)12.2(G)0(l)16.7(o)-369.5(Lu)20.9(ci)16.2(f)12.2(e)0(r)18.4(a)0(s)18.1(e)-372.9(as)18.1(sa)18.1(y)-371.1(s)0(y)19.6(s)0(t)17.2(e)12.7(m)-370.6(\()0(P)17.1(r)0(o)21.8(m)0(e)15.2(g)14.6(a\))-366.7(on)-360.2(a)-372.6(F)0(L)17.9(U)0(O)20.9(s)0(t)17.2(a)13.1(r)]TJ
0 -1.2203 TD
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T*
[(b)14.6(l)15.4(a)13.1(s)0(t)29.8(o)0(c)29.6(y)14.6(st)-425.4(g)14.6(r)0(o)28.1(u)0(p)-426.3(\()18.5(Ta)29.1(b)14.6(l)15.4(e)-442.5(3)19.5(\))12.2(.)-439.2(A)17.6(m)15.1(on)25.5(g)-440.7(t)18.5(h)15.6(o)0(s)27.5(e)-442.5(7)19.5(7)-442.1(m)15.1(i)15.4(R)14.7(N)12.7(A)17.6(s)0(,)-427.9(t)18.5(h)15.6(e)12.7(re)]TJ
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T*
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[(miR)-14.6(N)12.8(A)-452.3(w)2.7(ith)-447.9(a)-444.1(s)-14.6(i)12.2(ngle-nucleotide)-450.8(d)1.4(iffer)-14.6(e)10.1(nce)-444.5(b)0(etween)-447.9(the)-450.8(h)1(u)-14.6(-)]TJ
T*
0 Tc
[(m)15.1(a)13.1(n)-363.8(a)19.5(n)15.6(d)-363.4(t)12.2(h)15.6(e)-360.3(b)14.6(o)16.2(v)18.2(in)24.7(e)-360.3(m)15.1(i)15.4(R)14.7(N)12.7(A)-355.5(\()12.2(m)15.1(i)15.4(R)14.7(-)12.2(1)13.1(3)19.5(4)13.1(;)-357(S)14.2(up)28.9(pl)26.7(e)12.8(m)15.1(e)12.8(n)15.6(t)18.5(a)13.1(ry)-346.6(T)16(a)13.1(b)14.5(l)15.4(e)]TJ
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[(S)14.2(2)13.1(\))12.2(.)-401.3(T)16(w)17.3(e)12.7(l)15.4(ve)-386.3(m)15.1(i)15.4(R)14.7(N)12.7(A)17.6(s)-406(o)16.2(u)17.6(t)-405.1(o)16.2(f)-398.8(7)13.1(7)-404.2(w)17.3(e)12.8(r)18.3(e)-404.6(s)17.7(i)15.4(g)14.6(n)15.6(i)0()25.1(c)13.4(a)19.5(n)15.6(t)12.2(l)15.4(y)-402.8(d)16(i)15.4(f)12.2(f)18.5(e)12.8(re)24.7(n)15.6(t)18.5(ly)]TJ
28.0042 44.899 TD
[(e)12.7(x)16.8(p)17.6(r)0(e)24.7(s)17.7(se)30.4(d)-312.8(b)14.6(e)19.1(t)12.2(we)30.1(e)12.7(n)-306.9(e)12.7(a)13.1(r)18.3(l)0(y)-298.8(a)19.5(nd)-297.2(h)15.6(a)13.1(t)18.5(c)13.4(he)28.3(d)-312.8(b)14.6(l)15.4(a)19.5(st)23.5(o)16.2(c)13.4(y)14.6(s)17.7(t)12.2(s)17.7(.)-312.7(G)20.2(e)12.8(n)15.6(e)12.7(-)12.2(b)14.5(a)19.5(se)24.1(d)]TJ
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[(c)13.4(l)15.4(u)17.6(s)0(t)29.8(e)12.7(r)-437(a)19.5(na)28.7(l)15.4(y)14.6(si)26.8(s)-437.6(o)16.2(f)-436.8(t)18.5(h)15.6(e)12.7(se)-424.9(1)19.5(2)-435.8(m)15.1(i)15.4(R)14.7(N)12.7(A)17.6(s)-437.6(s)17.7(h)0(o)25.5(w)17.3(e)12.7(d)-433(t)12.2(w)17.3(o)-439.1(2)-429.5(c)13.4(l)15.4(us)28.9(t)12.2(e)12.7(r)18.3(s)]TJ
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T*
[(h)15.6(i)15.4(g)14.5(h)15.6(e)12.7(r)-278.9(e)12.7(x)16.8(pr)23.2(e)19.1(s)0(s)29(e)12.8(d)-281.2(i)15.4(n)-281.6(e)12.7(a)13.1(r)18.3(l)15.4(y)-282.6(b)14.5(l)15.4(a)13.1(s)17.7(t)12.2(o)16.2(c)13.4(y)14.5(st)29.8(s)-285.8(c)19.7(om)25(p)17.6(a)13.1(r)18.3(e)12.7(d)-281.2(t)12.2(o)-281(t)12.2(h)15.6(e)-278.1(ha)28.7(t)12.2(c)13.4(h)15.6(e)12.8(d)]TJ
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T*
-.0139 Tc
[(ca)5.6(ntly)-239.6(higher)-235.9(exp)-13.9(r)15.7(es)-13.9(s)15.1(e)-1.1(d)-238.2(i)1.5(n)-238.6(t)4.6(h)-13.9(e)-225.8(hatc)5.8(h)-13.9(e)14.4(d)-238.2(b).7(lastocys)-13.9(t)15.9(s)-242.8(c)5.8(o)-13.9(m)11.1(p)3.7(ar)4.4(ed)-238.2(to)]TJ
T*
-.0141 Tc
[(t)4.4(h)-14.1(e)-238.7(e)-1.3(a)5.4(r)-14.1(l)13.3(y)-252.5(bla)5.4(s)-14.1(t)9.4(o)2.1(cysts)-249.4(\()-1.9(hsa-miR-)4.4(127,)-244.6(h)-14.1(s)12.8(a-)4.4(mi)-14.1(R)9.7(-)4.4(130)5.4(a,)-251(hsa-miR-1)5.4(55,)]TJ
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[(hs)-14.3(a)16.5(-)-2.1(miR-1)5.2(96a,)-238.5(hs)-14.3(a)10.2(-)4.2(miR)-14.3(-)12.6(203)5.2(,)-244.8(h)1.3(s)-14.3(a)10.2(-)4.2(m).8(iR-28)5.2(,)-244.8(hs)-14.3(a)10.2(-)4.2(miR-29c)5.4(,)-244.8(a)-1.2(nd)-244.9(hs)-14.3(a)16.5(-)]TJ
0 -1.2203 TD
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[(miR-376a;)-333.6(M)-.6(annWhitney)-328.7(t)-2.3(est,)]TJ
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0 Tc
(P)Tj
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()Tj
/F4 1 Tf
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[(0)5.7(.)2.2(05;)-332.9(Fig.)-326.5(3\).)]TJ
/F8 1 Tf
9.9626 0 0 9.9626 310.8472 339.1369 Tm
0 Tc
[(Target)-336.8(gene)-341.3(predic)-7.4(tion,)-335.3(pathw)-8.2(ay)-336.5(analysis,)]TJ
0 -1.2007 TD
[(and)-335.7(expressi)-8.9(on)-335.7(analysi)-9.3(s)]TJ
/F4 1 Tf
8.9663 0 0 8.9663 320.8251 309.3164 Tm
[(For)-305.1(each)-305.6(miRNA,)-306.8(target)-309(genes)-306.1(were)-311.9(predicted)-308.3(using)-304.9(three)]TJ
-1.1128 -1.2203 TD
[(different)-271.1(programs.)-270.5(The)-271.8(results)-267.8(of)-268.8(the)-269.3(target)-271(gene)-273.2(predictions)]TJ
T*
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1.1128 -1.2266 TD
[(The)-290.8(union)-299(of)-294.1(the)-300.9(target)-296.3(gene)-298.5(lists)-294.7(was)-293.3(analyzed)-297.3(using)-298.5(IPA)]TJ
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[(software)-493.6(to)-490.1(search)-491(for)-490.8(enriched)-493.6(pathways)-492.1(and)-486.4(gene)-488.1(func-)]TJ
T*
[(tions.)-254.7(IPA)-257.8(analysis)-256.8(showed)-258.6(that)-256.8(the)-256.7(combined)-260.5(target)-258.4(gene)-254.2(list)]TJ
T*
[(was)-615.8(enriched)-613.8(for)-617.3(genes)-615.9(belonging)-617.8(to)-610.2(signal-transduction)]TJ
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[(pathways)-340.4(involved)-340.1(in)-342(the)-338.9(embryo)-338.6(development)-347.6(and)-334.7(regula-)]TJ
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[(tion)-395.9(of)-395.3(stem)-397.6(cell)-398.3(pluripotency,)-398(such)-397.4(as)-399.2(the)-395.8(TGF-)]TJ
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(b)Tj
/F4 1 Tf
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(signaling)Tj
-22.655 -1.2203 TD
[(pathway,)-285.1(WNT/)]TJ
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(b)Tj
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[(-catenin)-288.2(signaling)-286.3(pathway,)-291.4(and)-284.1(the)-288.3(ERK/)]TJ
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[(MAPK)-333.2(signaling)-336.9(pathways)-334.1(\(Table)-333.3(4\).)]TJ
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[(Next,)-378.5(a)-378.9(selection)-380.8(of)-376.3(target)-384.8(genes)-382(was)-375.5(made)-379.3(based)-374.9(on)-379.2(the)]TJ
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[(the)-269.3(experimentally)-266.4(supported)-271.5(miRNA-mRNA)-269.6(interactions)-271.2(as)]TJ
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[(reported)-495.7(by)-495.7(Tarbase)-495.2([41])-493.2(\(www.diana.pcbi.upenn.edu/tar-)]TJ
T*
[(base\),)-343.6(the)-345.2(IPA)-346.3(analysis,)-341.9(and)-347.3(the)-345.2(gene)-342.7(functions)-351.1(described)-343.8(in)]TJ
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[(literature.)-398.7(Using)-399.8(these)-403.3(selection)-399.8(criteria,)-402.6(a)-397.8(list)-400.9(of)-395.3(187)-396.9(candi-)]TJ
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[(date)-287.4(target)-296.3(genes)-293.4(for)-288.5(the)-294.6(12)-289.9(differentially)-291.9(expressed)-294.1(miRNAs)]TJ
T*
[(was)-331.2(established,)-339.3(to)-332(be)-333.1(validated)-335.7(by)-337.6(RT-qPCR.)]TJ
1.1128 -1.2203 TD
[(Gene)-590(expression)-598(analysis)-591.9(on)-587.9(eight)-593.4(samples)-592.5(per)-596.3(group)]TJ
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[(demonstrated)-429.3(that)-433.8(112)-428.5(of)-426.9(the)-427.4(187)-428.5(tested)-428.7(target)-435.4(genes)-426.2(were)]TJ
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T*
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[(0.05;)-491.2(Table)-497.2(5;)-495.6(Supplementary)]TJ
ET
q
360.926 0 0 242.419 59.754 491.584 cm
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BT
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[(FIG.)-899.5(1.)]TJ
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[(Overview)-901.7(of)-901.1(the)]TJ
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[(miR-218-binding)-511(sites)-506(in)-512.7(the)]TJ
T*
(3)Tj
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[(UTRs)-686.5(of)]TJ
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(NOG)Tj
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[(and)-587.6(of)-585(the)-585.5(miR-449b-)]TJ
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[(binding)-841.3(sites)-841.2(in)]TJ
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(NOTCH1)Tj
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(.)Tj
-12.7786 -1.1128 TD
[(The)-784(underlined)-780.4(nucleotides)]TJ
T*
[(were)-444.7(mutated)-451.2(in)-443.2(the)-446.4(mutant)]TJ
T*
(3)Tj
/F7 1 Tf
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[(UTR)-584(constructs.)-588.7(3)]TJ
/F7 1 Tf
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()Tj
/F4 1 Tf
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[(UTR,)-586.9(3)]TJ
/F7 1 Tf
3.3764 0 TD
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/F4 1 Tf
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[(untranslated)-339(region.)]TJ
/F1 1 Tf
6.4556 0 0 6.4556 59.7543 749.7069 Tm
(MICRO)Tj
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[(RNA)-337.2(EXPRESSION)-341.1(ANALYSIS)-333.8(IN)-335.1(BOVINE)-336.8(BLASTOCYSTS)-24066.1(5)]TJ
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