HP Custom siRNA

The number of transfections you can perform with 20 nmol HPP grade siRNA depends on the plate format used, the Transfection Reagent, and additional optimization requirements for your specific application.

In general, when using the HiPerFect Transfection Reagent in a 24-well plate format with 37.5 ng of siRNA per transfection you should be able to perform at least 6600 transfections.

If you are interested in other siRNA synthesis scales and purification options, please visit the QIAGEN Custom siRNA Synthesis page and our GeneGlobe data base, where you can find a large selection of pre-designed and validated siRNAs targeted at human, mouse and rat genes.

Yes. Below are a few selected references you can review:

• Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T. "Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate." EMBO J. 2001, 20 (23): 6877-88
• Zamore PD, Tuschl T, Sharp PA, Bartel DP.  "RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals." Cell. 2000, 101 (1): 25-33
• Williams RW, Rubin GM. "ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos." Proc Natl Acad Sci U S A. 2002, 99 (10): 6889-94

QIAGEN siRNA is delivered as a stable, ready-to-use duplex and does not need to be deprotected, desalted, quantified, or annealed before use. Simply resuspend the lyophilized RNA in the sterile RNAse-free water provided and transfect. Instructions for preparing your siRNA are provided on the data sheet supplied with each siRNA shipment.

Yes, Northern Blot Analysis has been shown in the literature to detect siRNA-induced reduction of specific mRNA. Whether a Northern Blot will be sensitive enough to detect a mRNA under investigation mainly depends on the expression level of the respective gene in the untreated control.

You can find an example for this application in the reference "Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems". Caplen et al., PNAS 2001, vol. 98, no. 17, pages 9742-9747.

We recommend to perform real-time RT-PCR for exact quantification of mRNA expression levels.

For the fixation of cells transfected with fluorescently labeled siRNA, we would suggest to perform the following protocol:

1. After transfection, remove the medium from the cells and wash the cells once with PBS.
2. Incubate the cells for 15 minutes at room temperature with 4% Paraformaldehyde (in PBS, pH 7.0). The cells should be completely covered by this solution (e.g., for a 96-well plate use 50 µl solution/well)
3. Wash the cells with PBS.

Fixed cells can be stored at 4°C for a few days.

(Note: It is also possible to use chamber slides or object slides for this procedure. Object slides should be coated to provide better growing conditions for cells. Cells can be fixed as described above and then overlayed with embedding medium to allow investigation using a fluorescence microscope. Optimal conditions for this method need to be determined by the user)

Labeling siRNA duplexes with Alexa Fluor dye or other dye labels at the 3’ end or the 5’ end of the sense strand does not influence gene silencing. We generally recommend the 3' end of the sense strand since new data suggests that a 3' end-labeled sense strand minimizes any chance of steric hindrance or interference with RNAi. Literature indicates that a free 5'-OH group on the antisense strand is required for the siRNA to be functional.

See the reference: Chiu Y L, and Rana T M, 2002.  RNAi in Human Cells: Basic Structural and Functional Features of Small Interfering RNA. Molecular Cell, 10, 549–561.

FlexiTube siRNA, FlexiTube GeneSolution, FlexiTube siRNA Premix, FlexiPlate siRNA, and GeneFamily Lists siRNAs can be ordered by catalog number over the telephone.

However, to ensure accuracy, Custom siRNA Synthesis orders should be submitted in writing. Therefore you can use the HP Custom siRNA Order Form https://www.qiagen.com/products/genesilencing/customsirna/customsirnaorder.aspx?EmailOrdering=1.

Visit the RNAi Solutions page http://www.qiagen.com/products/rnai/default.aspx?r=2714 on our homepage for access to the Online Ordering Tool, and choose the order link for your product of interest.

Here are examples of references that describe the inhibition of gene expression by siRNA in Xenopus and Zebrafish:

• Wargelius A, Ellingsen S, Fjose A. Double-stranded RNA induces specific developmental defects in zebrafish embryos.  Biochem Biophys Res Commun. 1999 Sep 16; 263(1): 156-61
• Zhou Y, Ching YP, Kok KH, Kung HF, Jin DY. Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.  Nucleic Acids Res. 2002 Apr 1; 30(7): 1664-9
• Zhao, Y Cao, M Li, and A Meng Double-stranded RNA injection produces nonspecific defects in zebrafish. Dev Biol, Jan 2001; 229(1): 215-23."