Methods for providing single-stranded RNA
A single-stranded, cellulose-based technology for use in DNA/RNA fragmentation, DNA preparation, recombinant DNA technology, etc., which can solve problems such as increasing complexity and cost, inducing undesired reactions, etc.
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Embodiment 1
[0277] Example 1 - Pulling down dsRNA from IVT RNA using cellulose
[0278] To test the feasibility of applying cellulose to remove dsRNA contaminants from IVT RNA, a simple pull-down experiment was first performed. 50 μg of 2,500 nt long N 1 - Methyl-pseudouridine (m1Ψ) modified IVT RNA was incubated with 0.1 g cellulose in the presence of 1×STE buffer containing 16% (v / v) EtOH. After centrifugation, unbound RNA in the supernatant was pelleted. Cellulose-bound RNA was recovered by resuspending the cellulose in 1× STE without EtOH, centrifuging and pelleting the supernatant. RNA from both fractions as well as the starting RNA material was analyzed for dsRNA content by dot blot using a dsRNA-specific J2 antibody. RNA integrity was monitored by agarose gel electrophoresis.
[0279] Dot blot analysis revealed that dsRNA content was greatly reduced in the unbound RNA fraction after incubation with cellulose compared to untreated input IVT RNA ( figure 1 ). This is due to th...
Embodiment 2
[0280] Example 2 - Effect of different concentrations of EtOH on the efficiency of removing dsRNA from IVT RNA by cellulose
[0281] In a next step, the purification method described above (see Example 1) was adapted to isolate unbound RNA from the cellulose using a microcentrifuge spin column. The advantage of this technique is the complete removal of liquid and thus unbound RNA from the cellulose by centrifugation. Furthermore, it was tested whether increasing the EtOH concentration up to 18% (v / v) or 20% (v / v) during IVT RNA incubation with cellulose would increase the efficiency of dsRNA removal. First, 50 μg of 1,500nt longpseudouridine (Ψ) was modified in the presence of 1×STE buffer containing 16% (v / v), 18% (v / v), or 20% (v / v) EtOH and D2-capped IVT RNA (prepurified from IVT reactions by magnetic beads) were incubated with 0.1 g cellulose in a microcentrifuge spin column. After centrifugation, unbound RNA was collected by centrifuging the column and pelleted. Cellul...
Embodiment 3
[0283] Example 3-Comparison of cellulose purification with RNaseIII treatment and HPLC purification
[0284]To test whether multiple cycles of cellulose purification using a microcentrifuge spin column according to the method described above (see Example 2) increased the efficiency of dsRNA removal, 1×STE with 16% (v / v) EtOH was used. Buffer, 100 μg of 2,500 nt long mlΨ-modified IVT RNA pre-purified by lithium chloride (LiCl) precipitation from the IVT reaction was purified 1x, 2x or 3x as described above. In addition, RNA purified by cellulose was compared to IVT RNA purified by E. coli RNase III (0.2 U / 100 μg RNA) for 30 min at 37° C. or by HPLC according to the protocol described by Weissman et al. (supra). All RNAs were analyzed for dsRNA content by dot blot using a dsRNA-specific J2 antibody and quantified by densitometric analysis of the hybridization signal. RNA integrity was monitored by agarose gel electrophoresis.
[0285] Increasing the number of cellulose purific...
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