Limits...
Single-pot enzymatic synthesis of Dicer-substrate siRNAs.

Guiley KZ, Pratt AJ, MacRae IJ - Nucleic Acids Res. (2011)

Bottom Line: The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25-27 nt RNA pools during in vitro transcription.The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells.The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.

ABSTRACT
We describe an inexpensive and efficient method for generating functional pools of Dicer-substrate small interfering RNAs (siRNAs) in a single reaction tube. The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25-27 nt RNA pools during in vitro transcription. The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells. The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.

Show MeSH
Silencing of a luciferase reporter by Giardia Dicer products. (A) HEK293 FT cells were transfected with firefly and Renilla luciferase reporter plasmids and increasing concentrations of siRNA pools targeting firefly luciferase. Small RNA pools generated by either Giardia or human Dicer enzymes were tested. Relative firefly luciferase activity levels (normalized to Renilla levels) are plotted is as a function of siRNA concentration. Data points are averages of three independent measurements ± standard deviation. (B) HEK 293 or HeLa cells were transfected with firefly and Renilla luciferase reporters and siRNA pools (200 ng/ml; 12–15 nM final concentration) targeting firefly luciferase that had been generated by human Dicer, Giardia Dicer, or bacterial RNase III. Relative firefly activity levels are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3299999&req=5

gkr1174-F3: Silencing of a luciferase reporter by Giardia Dicer products. (A) HEK293 FT cells were transfected with firefly and Renilla luciferase reporter plasmids and increasing concentrations of siRNA pools targeting firefly luciferase. Small RNA pools generated by either Giardia or human Dicer enzymes were tested. Relative firefly luciferase activity levels (normalized to Renilla levels) are plotted is as a function of siRNA concentration. Data points are averages of three independent measurements ± standard deviation. (B) HEK 293 or HeLa cells were transfected with firefly and Renilla luciferase reporters and siRNA pools (200 ng/ml; 12–15 nM final concentration) targeting firefly luciferase that had been generated by human Dicer, Giardia Dicer, or bacterial RNase III. Relative firefly activity levels are shown.

Mentions: We next tested the ability of Giardia Dicer siRNA pools to specifically silence a reporter gene when transfected into mammalian cells. Giardia Dicer was used to generate small RNAs against a 500 bp segment of the firefly luciferase gene. Various concentrations of the small RNAs were transfected with fixed amounts of firefly and Renilla luciferase reporter plasmids into HEK 293 cells. Renilla luciferase was included to normalize for transfection efficiency and also served as an indicator of non-specific gene silencing. Transfecting 4.5 nM Giardia Dicer small RNAs silenced more than 95% of the co-transfected firefly reporter compared to the no siRNA control (Figure 3A). Increasing the concentration of small RNAs to 45 nM silenced >98% of the firefly luciferase, with no appreciable effect on expression of Renilla. A pool of siRNAs generated by recombinant human Dicer, using the method of Myers et al. (2), produced a nearly identical dose response (Figure 3A), revealing that small RNA pools generated by Giardia Dicer have very similar silencing potencies to siRNA pools made by human Dicer. This observation is in contrast to the previous report that 27-nt DsiRNAs are more efficient at inducing RNAi than 21 nt siRNAs (20). Likewise, in a separate experiment, small RNAs generated by human Dicer, Giardia Dicer or RNase III gave rise to similar levels of luciferase silencing when transfected into either HeLa or HEK 293 cells (Figure 3B). Efficient silencing of firefly luciferase in NIH 3T3 cells (data not shown) also demonstrated that Giardia Dicer products can induce RNAi of a reporter gene in multiple mammalian cells types. The combined results suggest that the small RNAs produced by Giardia Dicer are functionally equivalent to small silencing RNAs generated using previously described methods.Figure 3.


Single-pot enzymatic synthesis of Dicer-substrate siRNAs.

Guiley KZ, Pratt AJ, MacRae IJ - Nucleic Acids Res. (2011)

Silencing of a luciferase reporter by Giardia Dicer products. (A) HEK293 FT cells were transfected with firefly and Renilla luciferase reporter plasmids and increasing concentrations of siRNA pools targeting firefly luciferase. Small RNA pools generated by either Giardia or human Dicer enzymes were tested. Relative firefly luciferase activity levels (normalized to Renilla levels) are plotted is as a function of siRNA concentration. Data points are averages of three independent measurements ± standard deviation. (B) HEK 293 or HeLa cells were transfected with firefly and Renilla luciferase reporters and siRNA pools (200 ng/ml; 12–15 nM final concentration) targeting firefly luciferase that had been generated by human Dicer, Giardia Dicer, or bacterial RNase III. Relative firefly activity levels are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299999&req=5

gkr1174-F3: Silencing of a luciferase reporter by Giardia Dicer products. (A) HEK293 FT cells were transfected with firefly and Renilla luciferase reporter plasmids and increasing concentrations of siRNA pools targeting firefly luciferase. Small RNA pools generated by either Giardia or human Dicer enzymes were tested. Relative firefly luciferase activity levels (normalized to Renilla levels) are plotted is as a function of siRNA concentration. Data points are averages of three independent measurements ± standard deviation. (B) HEK 293 or HeLa cells were transfected with firefly and Renilla luciferase reporters and siRNA pools (200 ng/ml; 12–15 nM final concentration) targeting firefly luciferase that had been generated by human Dicer, Giardia Dicer, or bacterial RNase III. Relative firefly activity levels are shown.
Mentions: We next tested the ability of Giardia Dicer siRNA pools to specifically silence a reporter gene when transfected into mammalian cells. Giardia Dicer was used to generate small RNAs against a 500 bp segment of the firefly luciferase gene. Various concentrations of the small RNAs were transfected with fixed amounts of firefly and Renilla luciferase reporter plasmids into HEK 293 cells. Renilla luciferase was included to normalize for transfection efficiency and also served as an indicator of non-specific gene silencing. Transfecting 4.5 nM Giardia Dicer small RNAs silenced more than 95% of the co-transfected firefly reporter compared to the no siRNA control (Figure 3A). Increasing the concentration of small RNAs to 45 nM silenced >98% of the firefly luciferase, with no appreciable effect on expression of Renilla. A pool of siRNAs generated by recombinant human Dicer, using the method of Myers et al. (2), produced a nearly identical dose response (Figure 3A), revealing that small RNA pools generated by Giardia Dicer have very similar silencing potencies to siRNA pools made by human Dicer. This observation is in contrast to the previous report that 27-nt DsiRNAs are more efficient at inducing RNAi than 21 nt siRNAs (20). Likewise, in a separate experiment, small RNAs generated by human Dicer, Giardia Dicer or RNase III gave rise to similar levels of luciferase silencing when transfected into either HeLa or HEK 293 cells (Figure 3B). Efficient silencing of firefly luciferase in NIH 3T3 cells (data not shown) also demonstrated that Giardia Dicer products can induce RNAi of a reporter gene in multiple mammalian cells types. The combined results suggest that the small RNAs produced by Giardia Dicer are functionally equivalent to small silencing RNAs generated using previously described methods.Figure 3.

Bottom Line: The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25-27 nt RNA pools during in vitro transcription.The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells.The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.

ABSTRACT
We describe an inexpensive and efficient method for generating functional pools of Dicer-substrate small interfering RNAs (siRNAs) in a single reaction tube. The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25-27 nt RNA pools during in vitro transcription. The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells. The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.

Show MeSH