Could anyone recommend a good siRNA negative control? Thanks a lot.
A non-silencing RNA duplex can be used as a control. I have seen this
5'-AAUUCUCCGAACGUGUCACGU-3' being used for HEK-293 cells.
Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1442-7.
Dharmacon makes several siRNA's for your specific target - and has a line of controls for sale also. They can be a non-targeting pool or a custom negative siRNA.
Check out the link for more info:
Ambion currently offers seven Silencer Negative Control siRNAs
Here is the General Information statement for the Silencer Negative Control #7siRNA - Cat#4644
"Small interfering RNA (siRNA) induced gene silencing in mammalian cells has shown great promise as a tool to suppress the expression of specific genes with high specificity (Elbashir et al. 2001). Ambions Silencer Negative Control siRNAs can be used to demonstrate that the transfection does not induce
nonspecific effects on gene expression. The Negative Control siRNAs are comprised of a 19 bp nontargeting sequence with 3 dT overhangs. The sequences have no significant homology to any known mouse, rat, or human gene sequences. The mRNA levels of housekeeping genes, including 28S rRNA, GAPDH, and cyclophilin, in transfected and nontransfected cells have been measured by Northern analysis using total RNA isolated 48 hr after transfection. The Negative Control siRNAs caused no gross changes in gene expression in the transfected cells, indicating a lack of nonspecific effects on gene expression. Several different Negative Control siRNAs are available from Ambion in the unlikely event that one has an
unanticipated effect on the expression pattern of the gene of interest.
The sense and antisense Negative Control siRNA strands are chemically synthesized, HPLC purified, and annealed. The siRNAs are ready to transfect and should be used at a final concentration of 1−100 nM. Enough Negative Control siRNA is provided to perform up to 300 transfections (using 15 pmol per well in a 24 well plate).
These siRNAs are susceptible to degradation by exogenous ribonucleases introduced during handling. As a precaution, your RNA oligonucleotides should not be handled with ungloved hands. RNase-free reagents, barrier pipette tips, and tubes should be used. Upon receipt, RNA oligonucleotides may be safely stored in a non-frost-free freezer for 6 months at or below 20°C. For long term storage, RNA oligonucleotides may be stored at or below 70°C."
Additionally, they offer the Silencer siRNA ScreeningControl Panel - Cat#4640 - a panel of all seven of their siRNA controls.
Here is the General Information statement for this product:
"Ambions Silencer siRNA Screening Control Panel contains seven Negative Control siRNAs that can be used to identify the best control for your cell system. Each Negative Control siRNA was designed to have no significant homology to any known gene sequences from human, mouse, or rat species. The Screening Control Panel enables RNAi researchers to test multiple Negative Control siRNAs to identify ones that do not induce nonspecific effects on gene expression in their chosen cell system.
In addition, an siRNA targeting Kif11 (Eg5) is included to provide an easy test for transfection efficiency. Knockdown of Kif11 leads to a cytotoxic response which can be visually assessed in many cell types. KIF11 encodes a motor protein that belongs to the kinesin-like protein family involved in chromosome positioning and bipolar spindle formation during cell mitosis. A reduction in KIF11 levels causes mitotic arrest. Cells treated with KIF11 siRNA appear rounded rather than flat, giving a visual indicator of successful siRNA delivery. Examples are available at www.ambion.com/info/kif11
The Negative Control siRNAs have been tested in human and rat cells and shown to have no significant impact on cell proliferation, apoptosis, or cell morphology. The KIF11 mRNA level in transfected and nontransfected cells has been measured by real-time RT-PCR using total RNA isolated 48 hr after transfection. KIF11 siRNA reduced the levels of KIF11 mRNA by 7095% in every cell line tested.
For each siRNA, the sense and antisense strands are chemically synthesized, HPLC purified, and then annealed. The annealed siRNAs are supplied dried, and Nuclease-free Water is provided for resuspension.
Thanks a lot for the helpful information!
How did you order the siRNA duplex product? From which company ? did you order as RNA oligo synthesis service?
Thanks for your help!