SilenCircleTM Basic RNAi Ex-
pression Kit
S ilenCircleTM RNAi system (pro-
tected under US patent 7,294,504
and additional patents pending) is
a plasmid-based RNA interference
system that uses engineered human
U6 RNApolymerase III promoter and
modified terminator for high level
small hairpin RNA (shRNA) or siRNA
expression inside mammalian cells.
The design enables precise start and
end of an interfering RNA with the
optimal 3’ overhanging nucleotides.
The pre-cut linear vector is ready-to-
ligate for construction of interfering
RNA expression plasmid with nearly
zero self-ligation. Bearing a neomycin
resistant marker, it may be used for
establishing siRNA-expressing stable
cell lines. SilenCircle RNAi system
has been successfully used to knock-
down expression of both endogenous
and exogenous genes.
Features
T he SilenCircletm Complete RNAi
kit is suitable for RNAi experi-
ments in tissue culture cells. Each
batch of reagents is vigorously tested
for consistency and stability, and offer
the following features:
- Efficient RNA interference
- Convenient ready-to-ligate format
- Almost no background ligation
- Without introducing extra bases
from restriction enzyme sites,
generate precise shRNA, siRNA, or
miRNA.
Materials Not Suppplied
with the Kit
-Target-specific insert DNA oligos
-E. coli competent cells
-Plasmid DNA purification system
-RNA purification system and reverse
transcription enzyme.
-Annealing buffer (10X)
-hcT4 DNA ligase with 10X buffer
-AvantGene
-DNA diluent
-5’ and 3’ p53 PCR primers
-5’ and 3’ Actin PCR primers
Allele Biotech-Introducing Cost Effectiveness to Research
Box 1 | Product Summary
Catalogue Numbers ABP-RI-SC02010
ABP-RI-SC02020
Components pre-cut pSIlenCircle Vector (10ng/µl)
pSilenCircle sequencing Primer (20µM)
p53-top (coding for p53 RNAi insert top strand)
(20µM)
p53-bot (coding for p53 RNAi insert bottom
strand) (20µM)
Storage All reagents to be stored at -20°C.
Stability All components are stable for 6 months when
stored properly.
Reagents Provided with Design of Inserts
the Kit
siRNA may be produced from two
T he kit provides enough reagents
to construct 10 RNAi expressing
plasmids, including Allele recom-
pSilenCircle plasmids encoding either
sense or antisense. Alternatively,
shRNA or miRNA may be produced
binant high concentration T4 DNA
ligase (hcT4 DNA ligase). DNA oligos
encoding each RNAi target sequence
from a single plasmid (shown below).
For each siRNA insert, two comple-
mentary synthetic DNA oligos are
need to be designed according to the
needed.
guidelines listed below.
Choose a target region that is A2N19
Oligos for generating p53-specific (sense sequence of the target RNA),
RNAi cassettes that have been tested design a linker sequence (e.g. 9
to significantly reduce p53 mRNA bases), use the following format:
levels are also included as positive
control.
5’ acacc N19 Linker N’19 t 3’
3’ g N’19 rcLinker N19 aaaaa 5’ #
The Complete Kit also provides
AvantGeneTM transfection reagent, “N’19” is reverse/complementary to
suitable for most mammalian cells “N19”; “rclinker” is reverse/ comple-
with exceptional efficiency. RT-PCR mentary to “linker”.
primers for detecting p53 and actin
mRNA levels are also included in the # Oligos orders are typically entered
Complete Kit. A successful p53 RNAi from 5’ to 3’, i.e. aaaaa N19...
experiment is expected to result in
reduced p53 mRNA levels while not Note: This product may be protected
affecting actin mRNA levels. RT-PCR under US patent 7,294,504 and additional
with p53 primers should generate a pending patents. Purchasing of this prod-
band of 496 bp; RT-PCR with actin uct grants the rights of use. Commercial
user may be required to obtain further
primers results in a band of 587b. license from third parties in order to use
certain RNA interference related technolo-
gies.
Protocols
Cloning
Oligo DNA annealing, ligation into the linearized vector, E.
coli transformation, and plasmid DNA preparation may be
performed according to standard protocols. Plasmid DNA
from positive clones will be cut only once by restriction
enzyme Stu I on the plasmid backbone (The Stu I site in
the Polylinker should have been deleted from the pre-cut
vector). A self-ligation plasmid (from the very few molecules
that escaped linearization of the vector) will yield two bands
of 1.1 kb and 3.5 kb, respectively. A sequencing primer is
also included in the kit for positive clone verification.
Transfection
Cells are prepared and transfected generally as you
would with a typical expression plasmid transfection. Most
commercial transfection reagents may be used with the
SilenCircleTM plasmids. Although using AvantGene trans-
fection reagent is recommended, in many cases the choice
of transfection reagent should depend on cells to be used.
Use 0.5 μg plasmid DNA per well of a 24-well plate as a
starting point.

Suggested Protocol for siRNA Insert Prepa-

F or Research Use Only. Not for
Diagnostic or Therapeutic Use.
Purchase does not include or carry
ration any right to resell or transfer this
product either as a stand-alone
product or as a component of another
Top oligo 1μg product. Any use of this product other
Bottom Oligo 1μg than the permitted use without the
express written authorization of Allele
Annealing Buffer (10X) 2μl
Biotech is strictly prohibited
Distilled Water 20μl

Heat at 95°C for 10min, slowly cool down to room tempera-

ture. Website: www.allelebiotech.com
Call: 1-800-991-RNAi/858-587-6645
(Pacific Time: 9:00AM~5:00PM)
Email: oligo@allelebiotech.com

Method Overview

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