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| Design | ||
| Synthesis | ||
| The Easiest Way to Get Them |
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Comparison
of Different siRNA Synthesis Methods
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| Chemical synthesis | In vitro transcription | Dicer/RNase III digestion | Expression vector | PCR expression cassette |
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Procedure
|
Order pre-designed siRNAs online
OR design siRNA and order custom siRNAs online |
Design siRNAs Order DNA oligos Perform IVT reaction Anneal Treat with DNase and ssRNA specific RNase Column purification |
Create transcription template
Perform IVT reaction Anneal Clean up dsRNA Digest with Dicer/RNase III Purification |
Design siRNAs Order DNA oligos Clone into cut vector Isolate clones Sequence verify Perform large scale prep |
Design
siRNAs Order DNA oligos Perform sequential PCR reactions Column purification |
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Hands on time
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None
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Moderate |
Moderate
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High |
Moderate
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Require-
ments |
Two 21mer RNA oligos
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Two 29mer DNA oligos |
Transcription
template (200-800 bp region flanked by promoters) |
Two 55-60mer DNA oligos |
Two ~50mer DNA oligos
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Need to
design siRNA? |
No (Ambion provides design)
|
Yes |
No
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Yes |
Yes
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Ability to fluorescently label siRNA
|
Yes
|
Yes |
Yes
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No |
No
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Relative ease of delivery
|
Good
|
Good |
Good
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Fair (viral vectors improve delivery in many systems) |
Fair
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Useful for
long-term studies? |
No
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No |
No
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Yes, using certain vectors with antibiotic resistance markers |
No
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Ability to
scale up synthesis |
Yes
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Limited |
Limited
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Yes |
Limited
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Ambion Product
|
Silencer siRNA Construction Kit |
pSilencer siRNA Expression Vectors pSilencer adeno 1.0-CMV System |
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Cost
(not including labor) |
$$$
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$$
|
$
|
$ |
$$
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