Detection of Small Nucleic Acids
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Materials and Methods
[0194]The following final concentrations (unless noted) were used for all reactions:[0195]Probe=1 μM[0196]INVADER=1 μM[0197]ARRESTOR=2.67 μM[0198]CLEAVASE XII enzyme=30 ng
All synthetic miRNA oligonucleotides were purchased from Dharmacon and gel purified on 20% denaturing acrylamide. Synthetic miRNAs were used to determine temperature optima (see below) and LOD.[0199]INVADER, probe, and ARRESTOR oligonucleotides were synthesized either by Integrated DNA Technologies (IDT) or Third Wave Technologies and purified on 20% denaturing acrylamide, unless otherwise indicated.[0200]The following 2.5× primary reaction buffer was used (unless otherwise noted) for all reactions:[0201]25 mM MOPS pH 7.5[0202]62.5 mM KCl[0203]0.125% Tween 20[0204]0.125% Nonidet NP40[0205]62.5 mM MgSO4 [0206]5% PEG[0207]Unless otherwise noted, all reactions were overlaid with 10 μl mineral oil prior to the first thermal incubation.[0208]Unless otherwise noted, synthetic miRNAs contained a 5′OH....
example 2
Temperature Optimization Experiments for let-7 and mir-1
[0209]The oligonucleotide design for let-7 is shown in FIG. 5. The oligonucleotide design for mir-1 is shown in FIG. 5. The following primary mixes were made and incubated at 50° C.±10° C. in a 96 well plate for 30 minutes. In addition, a no target master mix was prepared (addition of H2O in place of RNA). All reactions were covered with mineral oil to prevent evaporation.
StockAmountPrimary Reaction ComponentsConcentrationAddedPrimary Reaction Buffer2.5X4μlProbe oligonucleotide (SEQ ID NOs:10μM1μl2, 6, or 9 for let 7; SEQ ID NOs:12, 16, or 19 for miR-1)INVADER oligonucleotide (SEQ ID10μM1μlNOs: 1, 5, or 8 for let 7; SEQ IDNOs: 11, 15, or 18 for miR-1)CLEAVASE IX or XII enzyme40 ng / μl CLEAVASE0.5μlIX enzyme or60 ng / μl CLEAVASEXII enzymetRNA20ng / μl1.5μlSynthetic miRNA (SEQ ID NO: 4 for100pM2μllet-7a; SEQ ID NO: 14 for miR-1)Total10μl
[0210]After completion of the primary reaction, 5 μl of the following secondary reaction mix were ...
example 3
LOD Experiments for let-7 and miR-1
[0212]After determining the optimal reaction temperature for each set of probe and INVADER oligonucleotides and determining the best working design (from the temperature optimization net signal), the following experiment was set up to determine the LOD of the design using synthetic RNA. The following reaction mix was aliquoted into a 96-well plate (see plate setup below) with each well containing:
ComponentStock conc.Amount AddedPrimary reaction buffer2.5X4μlProbe10μM1μlSEQ ID NO: 6 for let 7SEQ ID NO: 16 or 19 for miR-1INVADER oligo10μM1μlSEQ ID NO: 5 for let 7SEQ ID NO: 15 or 18 for miR-1CLEAVASE XII enzyme60ng / μl0.5μlTRNA20ng / μl1μlTOTAL7.5μl
2.5 μl of the following miRNA concentrations were added in triplicates or quadruplicates using the following setup:
1 nM100 pM10 pM1 pM100 fM10 fMH2OABCD
The plate was overlayed with mineral oil (10 μl) and incubated at 50° C. for 2 hrs. After completion of the primary reaction, 5 μl of the following was added t...
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