HP Custom siRNA is an siRNA synthesis option that provides for specific siRNA requirements, including siRNA for multiple species, specific splice variants, and non-human, -mouse, and -rat genes.
HP Custom siRNA provides highly pure siRNA in 20 nmol amounts. Available fluorescent labels include Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 647, Cyanine 570, Cyanine 670, fluorescein, and rhodamine dyes. Modification options include amino linkers, thio linkers, and biotin, dabcyl, and phosphate modifications.
Successful RNAi experiments depend on effective delivery of siRNA into cells. Fluorescent dyes are widely used to label siRNA for transfection monitoring and optimization. HP Custom siRNA is available labeled with Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, or Alexa Fluor 647. Alexa Fluor dyes are more photostable and much brighter than traditionally used fluorescent dyes, making Alexa Fluor labeled HP Custom siRNA the ideal choice for monitoring transfection efficiency. Alexa Fluor labeled siRNA transfected into HeLa S3 cells shows 10–100 fold brighter fluorescence than fluorescein- and rhodamine-labeled siRNA (see figure " Alexa Fluor labeled siRNA provides brightest fluorescence"). The increased brightness and longer duration of Alexa Fluor dye fluorescence allow greater flexibility in RNAi optimization or cell-tracking experiments.
HP Custom siRNA is provided as High-Performance–Purity (HPP) grade. HPP-grade siRNA is the optimal siRNA purification grade for efficient gene silencing at an affordable cost. HPP-grade siRNA is synthesized using proprietary synthesis and purification processes, yielding siRNA that is >90% pure. There is no need for further time-consuming and expensive HPLC or PAGE purification. The high-throughput synthesis and purification processes enable fast turnaround times and high yields. Each siRNA duplex undergoes stringent quality control including MALDI-TOF mass spectrometry analysis. HPP Grade siRNA is economically priced allowing the use of highly pure siRNA in all routine RNAi experiments.
HP Custom siRNA provides purified, annealed, and desalted siRNA for effective gene silencing using your siRNA sequence of choice. Custom siRNA is available in individual tubes or 96-well plates. siRNA is provided as a stable duplex and there is no need to deprotect, desalt, quantify, or anneal before use. siRNA duplexes are provided as a lyophilized pellet to ensure stability during delivery. Before use, simply resuspend the lyophilized siRNA in the siRNA Suspension Buffer provided.
HP Custom siRNA is available with a variety of fluorescent labels and other modifications. Alexa Fluor dyes provide superior performance and can be directly substituted for other common dyes. Absorbance and emission maxima for available dyes, as well as dyes that can be replaced with spectrally similar Alexa Fluor dyes, are shown in the tables below. Other fluorescent dyes available include fluorescein, rhodamine, Cyanine 570, and Cyanine 670. HP Custom siRNA may be labeled at either the 3' or the 5' end of the sense strand, and labels do not affect the biological activity of the siRNA. As well as fluorescent labels, many other backbone and base modifications are available including amino linkers, thio linkers, and biotin, dabcyl, and phosphate modifications. Special modifications are also available on request; please contact QIAGEN Technical Service.
|Dye||Absorbance maximum (nm)||Emission maximum (nm)||Preferred replacement for:|
|Alexa Fluor 488||495||519||Fluorescein (FITC or FAM)|
|Alexa Fluor 546||556||573||Tetramethylrhodamine, Cyanine 570|
|Alexa Fluor 555||555||565||Cyanine 570|
|Alexa Fluor 647||650||665||Cyanine 670|
|Dye||Absorbance maximum (nm)||Emission maximum (nm)|
Highly pure siRNA can be used for a variety of applications, including:
Fluorescence microscopy of HeLa S3 cells 24 hours after transfection with 100 nM nonsilencing siRNA labeled at the 3' end of the sense strand with different fluorescent dyes.
|Target sequence provided||Already known|
|Scale or yield||20 nmol|
Please find a detailed description for the calculation of the silencing effect in QIAGEN News article 2006 e14 'Real-time RT-PCR for analysis of gene knockdown by RNAi - controls and calculations'.
Cells transfected with Alexa-Fluor labeled siRNA still show detectable fluorescence 72 hours after transfection. Certain Alexa dyes, e.g. Alexa Fluor 546, are detectable up to one week after transfection. By comparison, when labeling siRNA with Rhodamine or Fluorescein, transfected cells should be monitored for transfection efficiency after 3-4 hours.
Since Alexa Fluor dyes are more photostable, more resistant to variable pH conditions while in transit through the cell, and much brighter than traditionally used fluorescent dyes, Alexa Fluor labeled HPP Grade siRNA is the ideal choice for monitoring transfection efficiency.
For data and additional details on using fluorescently labeled siRNA, refer to QIAGEN News article e20, 2004: 'Alexa Fluor labeled siRNA is highly effective for monitoring transfection efficiency'.
The link below leads to a search tool run by TimeLogic Corporation:
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:
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:
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.
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: