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Yunke Song, Duncan Kilburn, Jee Hoon Song, Yulan Cheng, Christopher T. Saeui, Douglas G. Cheung, Carlo M. Croce, Kevin J. Yarema, Stephen J. Meltzer, Kelvin J. Liu, Tza-Huei Wang
Determination of absolute expression profiles using multiplexed miRNA analysis
www.plosone.org
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3.1578 0 Td
(tailor)Tj
2.3414 0 Td
(sensitivity,)Tj
-32.995 -1.3039 Td
(multiplex)Tj
4.0308 0 Td
(capability,)Tj
4.2916 0 Td
(and)Tj
1.7348 0 Td
(sample)Tj
3.0217 0 Td
(throughput)Tj
4.7849 0 Td
(based)Tj
2.4718 0 Td
(on)Tj
1.2756 0 Td
(application)Tj
4.6828 0 Td
(and)Tj
1.7291 0 Td
(detection)Tj
3.9401 0 Td
(methodology.)Tj
-31.9632 -1.2982 Td
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1.627 0 Td
(example,)Tj
3.7701 0 Td
(capillary)Tj
3.617 0 Td
(electrophoresis)Tj
6.2475 0 Td
(\(CE\))Tj
2.1316 0 Td
(and)Tj
1.7348 0 Td
(single)Tj
2.5285 0 Td
(molecule)Tj
3.8381 0 Td
(separation)Tj
4.3823 0 Td
([)Tj
0.83 0.64 0.02 0 k
(31)Tj
0 g
(])Tj
1.8539 0 Td
(may)Tj
1.9275 0 Td
(be)Tj
1.1509 0 Td
(used)Tj
-34.8092 -1.2983 Td
(instead)Tj
3.0784 0 Td
(of)Tj
1.0148 0 Td
(PAGE)Tj
2.7495 0 Td
(to)Tj
1.0262 0 Td
(further)Tj
3.0274 0 Td
(enhance)Tj
3.5489 0 Td
(multiplex)Tj
4.0309 0 Td
(capability)Tj
4.0705 0 Td
(and)Tj
1.7291 0 Td
(sensitivity.)Tj
-23.0795 -1.2983 Td
(Ligo-miR)Tj
4.0591 0 Td
(EZ's)Tj
1.9673 0 Td
(optimized)Tj
4.2576 0 Td
(2-step)Tj
2.6475 0 Td
(ligation)Tj
3.2542 0 Td
(mechanism)Tj
4.8415 0 Td
(enables)Tj
3.1635 0 Td
(high)Tj
2.0126 0 Td
(multiplex)Tj
4.0308 0 Td
(capability)Tj
4.0649 0 Td
(and)Tj
-35.4952 -1.3039 Td
(high)Tj
2.0069 0 Td
(specificity)Tj
4.2122 0 Td
(discrimination)Tj
6.1285 0 Td
(of)Tj
1.0148 0 Td
(closely-related)Tj
5.9527 0 Td
(miRNA)Tj
3.3505 0 Td
(species.)Tj
3.2202 0 Td
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4.3256 0 Td
(studies)Tj
2.9877 0 Td
(demon-)Tj
-33.1991 -1.2982 Td
(strate)Tj
2.4037 0 Td
(high)Tj
2.0069 0 Td
(specificity)Tj
4.2123 0 Td
(discrimination)Tj
6.1285 0 Td
(of)Tj
1.0147 0 Td
(miRNA)Tj
3.3506 0 Td
(family)Tj
2.7269 0 Td
(members)Tj
3.9458 0 Td
(with)Tj
2.0012 0 Td
(single)Tj
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(nucleotide)Tj
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(differ-)Tj
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(ences)Tj
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(and)Tj
1.7292 0 Td
(absolute)Tj
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(discrimination)Tj
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(of)Tj
1.0148 0 Td
(precursor)Tj
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(and)Tj
1.7291 0 Td
(mature)Tj
3.0954 0 Td
(miRNA.)Tj
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(A)Tj
0.9015 0 Td
(linear)Tj
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(amplification,)Tj
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(as)Tj
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(opposed)Tj
3.5773 0 Td
(to)Tj
1.0261 0 Td
(exponential)Tj
4.8982 0 Td
(amplification,)Tj
5.7657 0 Td
(is)Tj
0.8447 0 Td
(performed)Tj
4.473 0 Td
(during)Tj
2.9254 0 Td
(the)Tj
1.4683 0 Td
(2nd)Tj
1.7745 0 Td
(ligation)Tj
3.2541 0 Td
(step)Tj
1.8199 0 Td
(to)Tj
1.0261 0 Td
(enhance)Tj
-32.8533 -1.2983 Td
(sensitivity)Tj
4.2179 0 Td
(while)Tj
2.3697 0 Td
(minimizing)Tj
4.9039 0 Td
(amplification)Tj
5.5389 0 Td
(variability)Tj
4.2179 0 Td
(and)Tj
1.7348 0 Td
(bias)Tj
1.7858 0 Td
(for)Tj
1.389 0 Td
(high)Tj
2.0069 0 Td
(reproducibility)Tj
6.1965 0 Td
(and)Tj
-34.3613 -1.2983 Td
(high)Tj
2.0069 0 Td
(differential)Tj
4.6034 0 Td
(sensitivity.)Tj
4.4447 0 Td
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3.5773 0 Td
(of)Tj
1.0148 0 Td
(synthetic)Tj
3.8211 0 Td
(miRNA,)Tj
3.5773 0 Td
(cell)Tj
1.559 0 Td
(lines,)Tj
2.2904 0 Td
(and)Tj
1.7291 0 Td
(tissue)Tj
2.4605 0 Td
(samples)Tj
3.3845 0 Td
(show)Tj
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(that)Tj
1.7744 0 Td
(Ligo-miR)Tj
4.0592 0 Td
(EZ)Tj
1.389 0 Td
(is)Tj
0.8504 0 Td
(capable)Tj
3.2031 0 Td
(of)Tj
1.0148 0 Td
(20)Tj
1.1735 0 Td
(copies)Tj
2.7156 0 Td
(per)Tj
1.5364 0 Td
(cell)Tj
1.559 0 Td
(sensitivity)Tj
4.2179 0 Td
(with)Tj
2.0013 0 Td
(linearity)Tj
3.5263 0 Td
(across)Tj
2.6815 0 Td
(4.5)Tj
1.3947 0 Td
(orders)Tj
2.7836 0 Td
(of)Tj
-35.8807 -1.2982 Td
(magnitude.)Tj
4.7565 0 Td
(Analysis)Tj
3.5773 0 Td
(of)Tj
1.0148 0 Td
(mock)Tj
2.4661 0 Td
(expression)Tj
4.4674 0 Td
(panels)Tj
2.7495 0 Td
(demonstrates)Tj
5.5899 0 Td
(that)Tj
1.7802 0 Td
(differential)Tj
4.6034 0 Td
(sensitivity)Tj
4.2236 0 Td
(as)Tj
-35.2287 -1.2983 Td
(low)Tj
1.6554 0 Td
(as)Tj
/F11 1 Tf
0 Tc
1.0148 0 Td
(<)Tj
/F6 1 Tf
-0.004 Tc
(1.2)Tj
2.1713 0 Td
(fold)Tj
1.7858 0 Td
(is)Tj
0.8504 0 Td
(reliably)Tj
3.1691 0 Td
(achieved.)Tj
3.9288 0 Td
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1.1055 0 Td
(direct)Tj
2.5342 0 Td
(comparisons)Tj
5.3347 0 Td
(to)Tj
1.0262 0 Td
(microarray)Tj
4.6714 0 Td
(and)Tj
1.7348 0 Td
(RT-qPCR,)Tj
-30.9824 -1.3039 Td
(Ligo-miR)Tj
4.0535 0 Td
(EZ)Tj
1.3889 0 Td
(shows)Tj
2.6646 0 Td
(high)Tj
2.0069 0 Td
(correlations)Tj
4.9776 0 Td
(with)Tj
/F12 1 Tf
1.9956 0 Td
[()]TJ
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 374.5133 529.8519 cm
BT
/F6 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 378.085 525.3732 cm
BT
/F11 1 Tf
1 TL
0 0 Td
(>)Tj
/F6 1 Tf
-0.004 Tc
(0.9.)Tj
2.3924 0 Td
(Finally,)Tj
3.1691 0 Td
(we)Tj
1.3266 0 Td
(use)Tj
1.5364 0 Td
(Ligo-miR)Tj
4.0535 0 Td
(EZ)Tj
1.3889 0 Td
(to)Tj
1.0262 0 Td
(determine)Tj
-32.7002 -1.2982 Td
(miRNA)Tj
3.3392 0 Td
(copy)Tj
2.1146 0 Td
(number)Tj
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(in)Tj
1.0261 0 Td
(3)Tj
0.686 0 Td
(breast)Tj
2.6078 0 Td
(cell)Tj
1.5477 0 Td
(lines)Tj
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(and)Tj
1.7233 0 Td
(5)Tj
0.686 0 Td
(esophageal)Tj
4.5524 0 Td
(cell)Tj
1.5477 0 Td
(lines)Tj
2.058 0 Td
(and)Tj
1.7234 0 Td
(to)Tj
1.0148 0 Td
(quantify)Tj
3.5376 0 Td
(the)Tj
1.457 0 Td
(effects)Tj
-35.0869 -1.2983 Td
(of)Tj
1.0148 0 Td
(gemcitabine)Tj
5.0967 0 Td
(on)Tj
1.2756 0 Td
(metastatic)Tj
4.2746 0 Td
(pancreatic)Tj
4.3653 0 Td
(cancer)Tj
2.8402 0 Td
(cells.)Tj
2.1486 0 Td
(This)Tj
1.9843 0 Td
(system)Tj
2.9423 0 Td
(enables)Tj
3.1635 0 Td
(direct)Tj
2.5285 0 Td
(comparison)Tj
4.9776 0 Td
(of)Tj
-36.612 -1.2982 Td
(copy)Tj
2.1203 0 Td
(number)Tj
3.4186 0 Td
(expression)Tj
4.4674 0 Td
(profiles)Tj
3.1974 0 Td
(against)Tj
3.0274 0 Td
(relative)Tj
3.1407 0 Td
(expression)Tj
4.4673 0 Td
(profiles,)Tj
3.4243 0 Td
(through)Tj
3.4469 0 Td
(which)Tj
2.6475 0 Td
(we)Tj
1.3266 0 Td
(see)Tj
-34.6844 -1.304 Td
(that)Tj
1.7745 0 Td
(factors)Tj
2.9083 0 Td
(such)Tj
2.058 0 Td
(as)Tj
1.0148 0 Td
(cell)Tj
1.559 0 Td
(size)Tj
1.6838 0 Td
(and)Tj
1.7291 0 Td
(baseline)Tj
3.4299 0 Td
(transcription)Tj
5.4367 0 Td
(can)Tj
1.6271 0 Td
(contribute)Tj
4.388 0 Td
(significant)Tj
4.388 0 Td
(variability)Tj
4.2236 0 Td
(to)Tj
-36.2208 -1.2982 Td
(expression)Tj
4.4617 0 Td
(data.)Tj
-0.015 Tc
-3.2655 -1.2983 Td
(In)Tj
1.0885 0 Td
(addition)Tj
3.5093 0 Td
(to)Tj
1.0035 0 Td
(reducing)Tj
3.685 0 Td
(bias)Tj
1.7461 0 Td
(and)Tj
1.7008 0 Td
(variability)Tj
4.0988 0 Td
(in)Tj
1.0147 0 Td
(miRNA)Tj
3.2939 0 Td
(expression)Tj
4.354 0 Td
(profiles,)Tj
3.3278 0 Td
(measurement)Tj
5.5502 0 Td
(of)Tj
-35.5688 -1.3039 Td
(absolute)Tj
3.4356 0 Td
(expression)Tj
4.354 0 Td
(enables)Tj
3.0897 0 Td
(the)Tj
1.4343 0 Td
(determination)Tj
5.8109 0 Td
(of)Tj
0.9921 0 Td
(metrics)Tj
3.1068 0 Td
(such)Tj
2.0183 0 Td
(as)Tj
0.9921 0 Td
(accurate)Tj
3.4696 0 Td
(rank)Tj
2.0239 0 Td
(ordering)Tj
3.617 0 Td
(of)Tj
-34.3443 -1.2983 Td
(expression)Tj
4.354 0 Td
(and)Tj
1.6951 0 Td
(comparison)Tj
4.8699 0 Td
(of)Tj
0.9921 0 Td
(absolute)Tj
3.4356 0 Td
(changes)Tj
3.3277 0 Td
(in)Tj
1.0148 0 Td
(expression.)Tj
4.5695 0 Td
(These)Tj
2.5114 0 Td
(metrics,)Tj
3.3222 0 Td
(alongside)Tj
3.9005 0 Td
(the)Tj
1.4343 0 Td
(more)Tj
-35.4271 -1.2982 Td
(commonly)Tj
4.4561 0 Td
(measured)Tj
4.0195 0 Td
(differential)Tj
4.473 0 Td
(changes)Tj
3.3279 0 Td
(or)Tj
1.0771 0 Td
(proportional)Tj
5.1759 0 Td
(quantification,)Tj
5.8961 0 Td
(open)Tj
2.177 0 Td
(up)Tj
1.2415 0 Td
(an)Tj
1.1792 0 Td
(array)Tj
2.2337 0 Td
(of)Tj
-35.257 -1.304 Td
(new)Tj
1.8425 0 Td
(tools)Tj
2.0863 0 Td
(for)Tj
1.3493 0 Td
(comparing)Tj
4.4787 0 Td
(expression)Tj
4.354 0 Td
(profiles)Tj
3.1124 0 Td
(across)Tj
2.6134 0 Td
(patients,)Tj
3.4923 0 Td
(samples,)Tj
3.5206 0 Td
(and)Tj
1.6951 0 Td
(disease)Tj
2.9424 0 Td
(model)Tj
2.6872 0 Td
(systems.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 362.7212 cm
BT
/F2 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Materials)Tj
4.5071 0 Td
(and)Tj
1.989 0 Td
(methods)Tj
/F0 1 Tf
-6.4961 -1.5023 Td
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1.9134 0 Td
(line)Tj
1.748 0 Td
(samples)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 327.685 cm
BT
/F6 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Primary,)Tj
3.719 0 Td
(normal,)Tj
3.3732 0 Td
(non-immortalized)Tj
7.5684 0 Td
(esophageal)Tj
4.5638 0 Td
(epithelial)Tj
3.8664 0 Td
(cells)Tj
1.9219 0 Td
(\(HEEPIC\),)Tj
4.5921 0 Td
(along)Tj
2.4208 0 Td
(with)Tj
2.0012 0 Td
(esoph-)Tj
-34.0268 -1.2982 Td
(ageal)Tj
2.2223 0 Td
(cancer)Tj
2.846 0 Td
(cell)Tj
1.559 0 Td
(lines)Tj
2.0636 0 Td
(\(SKGT4)Tj
3.515 0 Td
(and)Tj
1.7291 0 Td
(OE33\),)Tj
3.05 0 Td
(were)Tj
2.126 0 Td
(purchased)Tj
4.3313 0 Td
(from)Tj
2.2054 0 Td
(ScienCell)Tj
3.9401 0 Td
(Research)Tj
3.8041 0 Td
(Laborato-)Tj
-33.3919 -1.2983 Td
(ries)Tj
1.644 0 Td
(\(Carlsbad,)Tj
4.3257 0 Td
(California,)Tj
4.473 0 Td
(USA\))Tj
2.4548 0 Td
(and)Tj
1.7291 0 Td
(Sigma)Tj
2.6816 0 Td
(Chemical)Tj
4.0252 0 Td
(\(St)Tj
1.3379 0 Td
(Louis,)Tj
2.6475 0 Td
(Missouri,)Tj
3.9969 0 Td
(USA\),)Tj
2.6815 0 Td
(respectively.)Tj
-31.9972 -1.3039 Td
(The)Tj
1.7801 0 Td
(Barrett's)Tj
3.5716 0 Td
(esophageal)Tj
4.5638 0 Td
(cell)Tj
1.559 0 Td
(lines)Tj
2.0636 0 Td
(\(CHTRT)Tj
3.8381 0 Td
(and)Tj
1.7291 0 Td
(QHTRT\))Tj
3.9175 0 Td
(were)Tj
2.1203 0 Td
(generous)Tj
3.8494 0 Td
(gifts)Tj
1.8992 0 Td
(of)Tj
1.0148 0 Td
(Dr.)Tj
1.5534 0 Td
(Peter)Tj
-33.4599 -1.2983 Td
(Rabinovitch,)Tj
5.3007 0 Td
(Fred)Tj
2.0693 0 Td
(Hutchinson)Tj
5.0003 0 Td
(Cancer)Tj
3.084 0 Td
(Center.)Tj
-14.2581 -1.2982 Td
(Breast)Tj
2.6929 0 Td
(cell)Tj
1.559 0 Td
(lines)Tj
2.0636 0 Td
(\(MCF-7,)Tj
3.6964 0 Td
(MCF-10A,)Tj
4.5127 0 Td
(and)Tj
1.7348 0 Td
(MDA-MB-231\))Tj
6.4686 0 Td
(and)Tj
1.7348 0 Td
(the)Tj
1.4683 0 Td
(metastatic)Tj
4.2747 0 Td
(pancreatic)Tj
4.3653 0 Td
(can-)Tj
-35.7673 -1.3039 Td
(cer)Tj
1.4343 0 Td
(cell)Tj
1.5647 0 Td
(line)Tj
1.7008 0 Td
(were)Tj
2.1259 0 Td
(purchased)Tj
4.3313 0 Td
(from)Tj
2.2054 0 Td
(ATCC)Tj
2.846 0 Td
(\(Manassas,)Tj
4.6091 0 Td
(VA\).)Tj
-19.6213 -1.2983 Td
(Details)Tj
2.9877 0 Td
(of)Tj
1.0148 0 Td
(cell)Tj
1.559 0 Td
(culturing)Tj
3.8948 0 Td
(protocols)Tj
3.9628 0 Td
(can)Tj
1.6271 0 Td
(be)Tj
1.1452 0 Td
(found)Tj
2.6192 0 Td
(in)Tj
0.83 0.64 0.02 0 k
1.0374 0 Td
(S1)Tj
1.1736 0 Td
(File)Tj
0 g
(.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 203.9811 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
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2.4189 0 Td
(RNA/Small)Tj
5.0598 0 Td
(RNA)Tj
2.315 0 Td
(fraction)Tj
3.4583 0 Td
(preparation)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 186.9732 cm
BT
/F6 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Total)Tj
2.313 0 Td
(RNA)Tj
2.262 0 Td
(was)Tj
1.7008 0 Td
(isolated)Tj
3.2825 0 Td
(from)Tj
2.211 0 Td
(the)Tj
1.4684 0 Td
(esophageal)Tj
4.558 0 Td
(cell)Tj
1.5591 0 Td
(lines)Tj
2.0693 0 Td
(using)Tj
2.3867 0 Td
(RNeasy)Tj
3.2485 0 Td
(kits)Tj
1.6441 0 Td
(\(Qiagen,)Tj
3.6737 0 Td
(Valencia,)Tj
-32.3771 -1.2982 Td
(CA\),)Tj
2.1316 0 Td
(combined)Tj
4.2406 0 Td
(with)Tj
1.9956 0 Td
(RNase-free)Tj
4.6544 0 Td
(DNase)Tj
2.9084 0 Td
(\(Qiagen,)Tj
3.668 0 Td
(#79254\),)Tj
3.6396 0 Td
(with)Tj
1.9956 0 Td
(TRIzol)Tj
2.9594 0 Td
(reagent)Tj
3.1804 0 Td
(\(Life)Tj
2.075 0 Td
(Technolo-)Tj
-33.4486 -1.2983 Td
(gies,)Tj
1.9558 0 Td
(Carlsbad,)Tj
3.9855 0 Td
(CA\))Tj
1.8992 0 Td
(used)Tj
2.058 0 Td
(instead)Tj
3.0727 0 Td
(of)Tj
1.0091 0 Td
(the)Tj
1.4627 0 Td
(QIAzol.)Tj
3.3789 0 Td
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1.6214 0 Td
(the)Tj
1.4627 0 Td
(RT-qPCR)Tj
4.1498 0 Td
(and)Tj
1.7235 0 Td
(microarray)Tj
4.6715 0 Td
(experiments,)Tj
-32.4508 -1.2982 Td
(this)Tj
1.6724 0 Td
(total)Tj
2.0069 0 Td
(RNA)Tj
2.262 0 Td
(was)Tj
1.6951 0 Td
(used)Tj
2.0636 0 Td
(directly.)Tj
3.4526 0 Td
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1.6271 0 Td
(Ligo-miR)Tj
4.0592 0 Td
(EZ)Tj
1.389 0 Td
(analysis,)Tj
3.5489 0 Td
(the)Tj
1.474 0 Td
(small)Tj
2.3244 0 Td
(RNA)Tj
2.262 0 Td
(fraction)Tj
3.3562 0 Td
(was)Tj
1.7008 0 Td
(iso-)Tj
-34.8942 -1.304 Td
(lated)Tj
2.1486 0 Td
(from)Tj
2.2053 0 Td
(this)Tj
1.6725 0 Td
(total)Tj
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