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HP Custom siRNA

For efficient gene silencing using high-purity siRNA

Features

  • High-purity siRNA at an economical price
  • Short turnaround times
  • Highly photostable and bright Alexa Fluor labels available
  • Option of a range of modifications

Products

The HP Custom siRNA is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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HP Custom siRNA with modifications

Cat. No. / ID: 1027424

Custom siRNA duplexes, available at different scales and with a range of modifications
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HP Custom siRNA without modifications

Cat. No. / ID: 1027423

Custom siRNA, 20 nmol, without modifications

Product Details

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.

Performance

Easy monitoring of transfection efficiency using Alexa Fluor dyes

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.

See figures

Principle

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.

Procedure

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.

A wide range of modification options

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.

Spectral characteristics of Alexa Fluor dyes
DyeAbsorbance maximum (nm)Emission maximum (nm)Preferred replacement for:
Alexa Fluor 488495519Fluorescein (FITC or FAM)
Alexa Fluor 546556573Tetramethylrhodamine, Cyanine 570
Alexa Fluor 555555565Cyanine 570
Alexa Fluor 647650665Cyanine 670
Spectral characteristics of the other available fluorescent dyes
DyeAbsorbance maximum (nm)Emission maximum (nm)
Cyanine 570550570
Cyanine 670649670
Fluorescein (FAM)494518
Tetramethylrhodamine (TAMRA)555580

Applications

Highly pure siRNA can be used for a variety of applications, including:

  • Functional genomics and proteomics research
  • Gene expression studies
  • Array analysis
  • Monitoring transfection efficiency and cell-tracking experiments

Supporting data and figures

Specifications

FeaturesSpecifications
DesignBy customer
Target sequence providedAlready known
Scale or yield20 nmol
SpeciesAll
FormatTube
Guarantee/validationNo guarantee
ModificationYes

Resources

Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.

FAQ

How do I calculate the percentage of silencing with real-time RT-PCR for siRNA?

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'.

 

FAQ ID -498
I want to start with gene silencing (RNAi) experiments. Do you have informational literature?
The QIAGEN e-News article: "Getting started in RNAi research" will provide useful information. You can access it at the following link: Getting started in RNAi research.
FAQ ID -580
How long is fluorescence detectable in cells after transfection with fluorescently labeled siRNAs?

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'.

FAQ ID -392
How are siRNAs introduced into C. elegans?
Two methods have been used; soaking worms in siRNA is successful and worms can be fed bacteria expressing the siRNA (from a plasmid after transformation). REFERENCES: RNAi in C. elegans: Soaking in the Genome Sequence Hiroaki Tabara, Alla Grishok, and Craig C. Mello 1998. Science 282:430. RH Plasterk and RF Ketting The silence of the genes. Curr Opin Genet Dev, Oct 2000; 10(5): 562-7.
FAQ ID -396
Where can I find the public Smith-Waterman homology search tool that you refer to on your siRNA online design page?

The link below leads to a search tool run by TimeLogic Corporation:

 Smith-Waterman homology search

FAQ ID -562
How many transfections can I perform with 20 nmol of HPP grade 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.

FAQ ID -367
Can siRNA silence bacterial genes?
RNA interference is unique to eukaryotes. Homologues to the genes involved in RNAi have not been found in the genomes of bacteria or archaea. Additionally, prokaryotes express RNase III, a very potent and fast-acting RNase that degrades dsRNA substrates as short as 12 bp.
FAQ ID -395
Do we recommend pooling siRNA sequences?
We do not recommend pooling siRNA sequences since this can result in more non-specific and off-target effects diluting the knockdown effect of the silencing siRNA.
FAQ ID -517
Can RNAi experiments be performed in Drosophila?

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

 

 

FAQ ID -436
Do I need to anneal, deprotect or desalt my QIAGEN siRNA?

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.

FAQ ID -398
Are Northern Blots sensitive enough to detect siRNA-induced gene silencing?

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.

FAQ ID -403
Do you have a protocol for the fixation of cells transfected with fluorescently labeled siRNA?

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)

 

FAQ ID -793
Where should I add a dye-label modification on the siRNA?

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.

 

FAQ ID -648
Can I use my own siRNA design?
Yes. If you provide the siRNA target sequence, we will synthesize, the corresponding HP Custom siRNA for you. Unfortunately, we do not offer custom designed and synthesized SureSilencing shPlasmids.
FAQ ID -2897
Where can I find QIAGEN products for a specific gene or gene product?
You can search for specific gene products in the QIAGEN GeneGlobe Database. This easy-to-use, comprehensive Web portal allows you to find information about, search for, and order high-quality products for human, mouse, and rat genes. QIAGEN provides a vast range of gene-specific products covering every aspect of an experiment, from gene silencing to expression analysis at the mRNA or protein level.
FAQ ID -803
How do I submit a siRNA order by telephone or online?

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.

FAQ ID -399
What is the composition of the siRNA resuspension & annealing buffer?
The composition of the siRNA resuspension & annealing buffer is 100 mM Potassium Acetate, 30 mM HEPES-KOH, pH 7.4.
FAQ ID -522
Has RNAi been successful using siRNA in Zebrafish and Xenopus?

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

FAQ ID -400
What is the average molecular weight of a siRNA, and how do I convert uM to ug values?
The Molecular Weight (MW) of a 21 nucleotide double-stranded siRNA molecule is approximately 13-15 ug/nmol. The exact MW depends on the sequence of the siRNA. 20 uM of double-stranded 21 nt siRNA is equivalent to approximately 0.25 ug/ul.
FAQ ID -388