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Felipe M. Vigoder, Darren J. Parker, Nicola Cook, Océane Tournière, Tanya Sneddon, Michael G. Ritchie
Inducing Cold-Sensitivity in the Frigophilic Fly Drosophila montana by RNAi
www.plosone.org
www.plosone.org
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(\(dsRNA\).)Tj
4.0535 0 Td
(Synthesis)Tj
3.9401 0 Td
(of)Tj
1.0148 0 Td
(dsRNA,)Tj
3.3732 0 Td
(using)Tj
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(T7)Tj
1.304 0 Td
(PCR)Tj
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(products)Tj
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(as)Tj
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(a)Tj
0.6519 0 Td
(template,)Tj
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(was)Tj
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(out)Tj
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(the)Tj
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(MEGAscript)Tj
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(T7)Tj
1.3096 0 Td
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(\(Life)Tj
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2.0352 0 Td
(Paisley,)Tj
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(UK\))Tj
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(the)Tj
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(man-)Tj
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(ufacturer's)Tj
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(instructions.)Tj
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(RNA)Tj
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(purified)Tj
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(Kit)Tj
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(\(Life)Tj
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(Ltd.,)Tj
2.0352 0 Td
(Paisley,)Tj
3.1918 0 Td
(UK\),)Tj
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(eluted)Tj
2.6702 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.652 0 Td
(low-salt)Tj
3.3448 0 Td
(buffer,)Tj
2.8517 0 Td
(and)Tj
1.7291 0 Td
(quantified)Tj
4.3086 0 Td
(using)Tj
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(a)Tj
0.6519 0 Td
(Nanodrop)Tj
-32.5868 -1.2982 Td
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(\(Thermo)Tj
3.8154 0 Td
(Fisher)Tj
2.6986 0 Td
(Scientific,)Tj
4.1329 0 Td
(Loughborough,)Tj
6.4233 0 Td
(UK\).)Tj
2.194 0 Td
(We)Tj
1.61 0 Td
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(dsRNA)Tj
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-35.9997 -1.2983 Td
[()39()6()7()]TJ
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1.8708 0 Td
(and)Tj
1.7291 0 Td
(also)Tj
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(the)Tj
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(bacterial)Tj
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(gene)Tj
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2.0806 0 Td
[()4( )6()]TJ
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1.8935 0 Td
(which)Tj
2.6476 0 Td
(was)Tj
1.7008 0 Td
(used)Tj
2.0579 0 Td
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(a)Tj
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(control.)Tj
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0.83 0.64 0.02 0 k
1.0375 0 Td
(S1)Tj
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0 g
(.)Tj
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Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 478.0346 cm
BT
/F0 1 Tf
1 TL
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6.2504 0 Td
(procedure)Tj
ET
Q
q
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1 J
0 w
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BT
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1 TL
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(flies)Tj
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1 J
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1 TL
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q
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1 J
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(injected)Tj
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(100)Tj
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(injected)Tj
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(the)Tj
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(a)Tj
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(207)Tj
1.5931 0 Td
(nl)Tj
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(of)Tj
0.9751 0 Td
(dsRNA)Tj
3.0614 0 Td
(\(4)Tj
/F16 1 Tf
0.9978 0 Td
[()]TJ
/F10 1 Tf
(g/)Tj
/F16 1 Tf
[()]TJ
/F10 1 Tf
(l\),)Tj
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(of)Tj
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(the)Tj
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(100)Tj
1.5931 0 Td
(flies)Tj
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(injected)Tj
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1.9218 0 Td
[()4( )6()]TJ
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1.8254 0 Td
(dsRNA.)Tj
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(was)Tj
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(a)Tj
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(Drummond)Tj
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(Nanoject)Tj
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(II)Tj
0.8561 0 Td
(microinjector)Tj
5.4652 0 Td
(\(Drummond)Tj
5.2439 0 Td
(Scientific)Tj
3.7134 0 Td
(Company,)Tj
4.2406 0 Td
(Broo-)Tj
-35.3534 -1.2982 Td
(mall,)Tj
2.0976 0 Td
(USA\).)Tj
2.5966 0 Td
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(injection,)Tj
3.804 0 Td
(individuals)Tj
4.4504 0 Td
(were)Tj
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(separated)Tj
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1.7688 0 Td
(small)Tj
2.2336 0 Td
(glass)Tj
2.0012 0 Td
(vials)Tj
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(containing)Tj
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(malt)Tj
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(food)Tj
-35.2343 -1.2983 Td
(and)Tj
1.6724 0 Td
(transferred)Tj
4.4447 0 Td
(to)Tj
0.9865 0 Td
(the)Tj
1.4173 0 Td
(appropriate)Tj
4.6828 0 Td
(incubator)Tj
3.9401 0 Td
(to)Tj
0.9865 0 Td
(assess)Tj
2.4151 0 Td
(their)Tj
2.0183 0 Td
(capacity)Tj
3.3277 0 Td
(to)Tj
0.9921 0 Td
(cold)Tj
1.8482 0 Td
(acclimate)Tj
3.8324 0 Td
(\(see)Tj
1.6951 0 Td
(below\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 354.0472 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Cold)Tj
2.2441 0 Td
(acclimation)Tj
5.1591 0 Td
(trials)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 337.0393 cm
BT
/F10 1 Tf
1 TL
-0.004 Tc
0 0 Td
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3.4582 0 Td
(flies)Tj
1.8085 0 Td
(were)Tj
2.1203 0 Td
(divided)Tj
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(into)Tj
1.8369 0 Td
(two)Tj
1.7121 0 Td
(groups,)Tj
3.1918 0 Td
(each)Tj
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(containing)Tj
4.5297 0 Td
(approximately)Tj
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(70)Tj
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(and)Tj
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(70)Tj
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(control)Tj
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(flies.)Tj
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(group)Tj
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(maintained)Tj
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(at)Tj
0.9524 0 Td
(the)Tj
1.4683 0 Td
(control)Tj
3.1238 0 Td
(temperature)Tj
5.1307 0 Td
(of)Tj
1.0148 0 Td
(19C)Tj
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(and)Tj
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(the)Tj
1.4684 0 Td
(second)Tj
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(at)Tj
-36.3115 -1.2982 Td
(5C)Tj
1.7517 0 Td
(\(22:2)Tj
2.2111 0 Td
(L:D\))Tj
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(fresh)Tj
2.1884 0 Td
(vials)Tj
1.9842 0 Td
(contain-)Tj
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(agar)Tj
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(\(10%\))Tj
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(moisture)Tj
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(and)Tj
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(a)Tj
0.652 0 Td
(cold)Tj
1.9275 0 Td
(shock:)Tj
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(-7C)Tj
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(for)Tj
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(16)Tj
1.1679 0 Td
(hours)Tj
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(constant)Tj
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(then)Tj
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(immediately)Tj
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1.0262 0 Td
(individual)Tj
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(plastic)Tj
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(containers)Tj
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(observation.)Tj
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(Chill-)Tj
-35.0189 -1.3039 Td
(coma)Tj
2.4037 0 Td
(recover)Tj
3.1918 0 Td
(time)Tj
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(\(CCRT\))Tj
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(a)Tj
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(measure)Tj
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(cold)Tj
1.9218 0 Td
(tolerance)Tj
3.8948 0 Td
(\(see)Tj
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(Vesala)Tj
/F15 1 Tf
2.812 0 Td
[()8()]TJ
0.9127 0 Td
[( )11()]TJ
/F10 1 Tf
(.)Tj
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(2012a\).)Tj
3.118 0 Td
(A)Tj
0.9071 0 Td
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(recovered)Tj
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(once)Tj
2.126 0 Td
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(all)Tj
1.1452 0 Td
(six)Tj
1.3153 0 Td
(legs.)Tj
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1.99 0 Td
(experiment)Tj
-33.0064 -1.2983 Td
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(blindly)Tj
3.0217 0 Td
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1.0205 0 Td
(minimise)Tj
3.9968 0 Td
(observer)Tj
3.6397 0 Td
(bias.)Tj
2.0125 0 Td
(Mortality)Tj
3.9912 0 Td
(rate)Tj
1.7461 0 Td
(after)Tj
2.0353 0 Td
(the)Tj
1.4683 0 Td
(3)Tj
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(days)Tj
1.9956 0 Td
(of)Tj
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(acclimation)Tj
-31.1695 -1.3039 Td
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1.4683 0 Td
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1.9276 0 Td
(shock)Tj
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1.7008 0 Td
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(A)Tj
0.9014 0 Td
(total)Tj
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(injected)Tj
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(the)Tj
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(experiment)Tj
-31.9009 -1.2983 Td
(divided)Tj
3.2144 0 Td
(equally)Tj
3.0614 0 Td
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1.0318 0 Td
(four)Tj
1.9162 0 Td
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(\(see)Tj
1.7688 0 Td
(below\).)Tj
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Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 190.9984 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Expression)Tj
5.1071 0 Td
(analyses)Tj
ET
Q
q
1 j
1 J
0 w
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BT
/F10 1 Tf
1 TL
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4.0138 0 Td
(PCR)Tj
2.0352 0 Td
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3.3732 0 Td
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1.7405 0 Td
(dsRNA)Tj
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(injections)Tj
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(produced)Tj
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(change)Tj
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(in)Tj
1.0205 0 Td
(the)Tj
-33.9475 -1.2982 Td
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1.44 0 Td
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2.4548 0 Td
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(analyses)Tj
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-35.5178 -1.2983 Td
(19C)Tj
2.1883 0 Td
(due)Tj
1.6667 0 Td
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1.0035 0 Td
(high)Tj
1.9672 0 Td
(mortality)Tj
3.8268 0 Td
(in)Tj
1.0204 0 Td
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(5C)Tj
1.7234 0 Td
(treatment)Tj
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(groups)Tj
2.9083 0 Td
(\(see)Tj
0.83 0.64 0.02 0 k
1.7291 0 Td
(results)Tj
0 g
(\).)Tj
3.2825 0 Td
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1.9956 0 Td
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-35.5689 -1.3039 Td
(19C)Tj
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1.3549 0 Td
(two)Tj
1.6781 0 Td
(weeks)Tj
2.5569 0 Td
(as)Tj
0.9978 0 Td
(per)Tj
1.5023 0 Td
(the)Tj
1.44 0 Td
(standard)Tj
3.6397 0 Td
(fly)Tj
1.1792 0 Td
(rearing)Tj
3.033 0 Td
(protocol.)Tj
3.7304 0 Td
(Approximately)Tj
6.1001 0 Td
(40)Tj
1.1509 0 Td
(females)Tj
3.1464 0 Td
(were)Tj
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(then)Tj
-35.7786 -1.2983 Td
(injected)Tj
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(with)Tj
1.9616 0 Td
(target)Tj
2.4491 0 Td
(dsRNA)Tj
3.1011 0 Td
(and)Tj
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(another)Tj
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(40)Tj
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/F15 1 Tf
1.9559 0 Td
[()4( )6()]TJ
/F10 1 Tf
1.8539 0 Td
(dsRNA.)Tj
3.3165 0 Td
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2.5172 0 Td
(females)Tj
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(transferred)Tj
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-36.3285 -1.2982 Td
(new)Tj
1.8425 0 Td
(vials)Tj
1.9332 0 Td
(containing)Tj
4.4277 0 Td
(malt)Tj
1.9785 0 Td
(food)Tj
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(and)Tj
1.7008 0 Td
(incubated)Tj
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0.9298 0 Td
(19C)Tj
2.1883 0 Td
(for)Tj
1.355 0 Td
(24)Tj
1.1508 0 Td
(hours.)Tj
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2.2677 0 Td
(RNA)Tj
2.2337 0 Td
(was)Tj
1.6667 0 Td
(extracted)Tj
-32.4564 -1.3039 Td
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2.1656 0 Td
(3)Tj
0.686 0 Td
(pools)Tj
2.3074 0 Td
(of)Tj
0.9921 0 Td
(10)Tj
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