banner: Custom siRNA Synthesis  

Custom siRNA Synthesis

siRNA customization has never been easier

No pre-designed product to fit your needs? Use our online design tools and extensive synthesis options to create a custom siRNA specific for your application. Numerous combinations of modifications, sizes, and purification options are available for convenient online ordering.

  • Preclinical scale and OEM RNA & DNA synthesis

    Preclinical scale/OEM service for synthesis of RNA and DNA at larger quantities

  • Order custom siRNA

    Order both modified or unmodified siRNAs. Patented modification patterns (ON-TARGET, ON-TARGETplus, siSTABLE, or Accell) and additional custom modifications are available.

  • Custom SMARTpool design & synthesis

    To complement our pre-designed siRNA collections and support a broad range of RNAi experiments, our siRNA design experts can provide custom SMARTpool reagents targeting genes outside of our genomewide offerings

  • siDESIGN Center

    Design siRNAs targeting genes in non-standard species, particular splice variants or homologous regions across gene families or species.

siRNA Yield Table

For unmodified siRNA, the following approximate yields can be expected:

  Standard (A4) Purified in vivo in vivo HPLC
nmol mg nmol mg nmol mg nmol mg
0.025 µmol scale 20 0.25 - - - - - -
0.05 µmol scale 40 0.5 20 0.25 25 .3 - -
0.2 µmol scale 150 2 80 1 100 1.3 50 0.65
0.4 µmol scale 300 4 160 2 200 2.6 100 1.3
1.0 µmol scale 750 10 320 4 500 6.6 250 3.3
2.0 µmol scale 1500 20 750 10 1000 13 500 6.6
5.0 µmol scale 3750 50 1875 25 2500 33 1250 16
10.0 µmol scale 7500 100 3750 50 5000 66 2500 33

Expert siRNA design assistance is available

Our capabilities include a range of alternative siRNA designs to empower your RNAi research

If you are working in human, mouse or rat models, we may already have what you need as a pre-designed product: Search for your gene in the Search field found in the upper right corner.

Take advantage of our internal design experts and SMARTpool technology! Order a functionally guaranteed Custom SMARTpool for worry-free customization.

Alternative siRNA designs that we support include:

  • Blunt-ended
  • Asymmetrical
  • Longer duplexes (>23 nt)
  • Mismatched strands
  • Alternative bases or linkages

If you can't find what you need or you require additional specifications not available through online order, please request a quote.

For further assistance, please contact Technical Support.

Proprietary siRNA

Modified for specificity, stability or self-delivery, we have the siRNA solution.

Choose one of our specialized chemical modification patterns to enhance your custom siRNA.

All siRNA modifications, proprietary and other, are available through custom siRNA synthesis  

  ON-TARGET ON-TARGETplus Accell siSTABLE
Inhibits sense (passenger) strand uptake by RISC
Antisense strand seed region modified for greater specificity to target      
Resistant to endo-and exonuclease degradation    
Delivery into cells without transfection reagent      
Also available as Pre-designed siRNA    

Dye Labels

Dharmacon offers a broad portfolio of dye modifications for fluorescent labeling of your custom siRNA and RNA oligonucleotides.

  • Include a fluorescent tag at the 3' or 5' positions on either strand of an siRNA
  • Synthesis of most dye-labeled siRNAs is scalable to gram quantities

Fluorescent dyes available online:

*Some 3' dye labels are only available upon request.
Fluorophore λ max abs (nm) λ max em (nm) Comparable to 5' or 3'
Fluorescein / 6-FAM 494 520   - both*
DY547 548 562   Cy3, Alexa 546, Alexa 555 both*
TAMRA 565 580   - both*
DY647 645 662   Cy5, Alexa 647 both*
DY677 684 698   CY5.5, Alexa 680 both*
Cy3 547 563   - both*
Cy5 646 662   - both*
Cy5.5 688 707   - both*

Some of the dye options listed in the above tables will produce higher oligo yields than others. For assistance in choosing the most appropriate dye label for your application, please contact Technical Support. You can also request a quote online if you already know that an alternative dye will be required for your experiments.

Alexa Fluor® is a registered trademark of Invitrogen Corporation. 

Chemical modifications available for custom siRNA

In addition to fluorescent dyes, we offer a broad portfolio of other chemical modifications that can also be applied to both siRNA and single-strand RNA.

For information on our specialized modification patterns for reducing off-targets, self-delivery and nuclease resistance, view the Proprietary siRNA tab.

siRNA can be modified at either end, on either strand. Multiple modifications are also available but availability may be dependent upon sequence or other factors. Please contact Technical Support for more information

 
 

Standard modifications for single-strand RNA can also be applied to siRNA by request. Please contact Technical Support to inquire about additional modifications or options for your siRNA, or request a quote now.

Purification & Processing

We routinely achieve 80-85% purity for unmodified siRNA strands without additional purification.

However, purification may be recommended when chemically synthesized siRNAs are:

  • Chemically modified at the 3' end or internally
  • Dually modified on the same strand
  • Intended for use in highly sensitive assays or in vivo applications

Please contact Technical Support for information regarding recommendations for purification or purity estimates for unpurified material.

siRNA Processing Options:

  Single Strands (A1) Standard (A4) HPLC In vivo In vivo HPLC
Desalted  
Deprotected  
Duplexed  
Purified      
Endotoxin tested      
Sodium counter-ion exchange      
Recommended for modified siRNA (dyes, etc.)      
Recommended for in vivo use      
2'ACE protected single-strands        

Description of siRNA Processing Terms

  • Desalted: The siRNA duplex has been desalted by either ethanol precipitation or C18 column desalting
  • Deprotected: The 2'-ACE protecting groups of the RNA bases have been removed (deprotected)
  • Duplexed: The two complementary siRNA strands have been annealed to form a duplex
  • Single-strand (A1): The siRNA is provided with the sense and antisense strands in separate tubes; individual strands have NOT been desalted or deprotected
  • Standard (A4): The siRNA is provided as a desalted & deprotected duplex, ready to use upon resuspension
  • HPLC: The duplex has undergone ion exchange High Performance Liquid Chromatography for purification
  • In vivo: The duplex has been processed by counter-ion (Na+) exchange, desalting, sterile filtration, and endotoxin testing
  • In vivo HPLC: The duplex has undergone both in vivo processing as well as HPLC purification
 

In vivo RNAi

Partner with the industry leader in RNAi technologies for the highest quality in vivo -ready RNAi reagents.

We want to ensure your in vivo experiments have the best chance of success. To assist you in your experimental planning, we offer the following options and guidance:

Assess your siRNA sequences in vitro

  • We recommend identifying a highly functional siRNA design in vitro prior to initiating expensive experiments using animal models
  • A predesigned Set of 4 siRNA reagents is ideal for testing multiple siRNA sequences
    • Individual duplexes: Four individual siRNAs are pre-designed for every gene in human, mouse and rat. Order 1, 2, 3 or 4.
    • SMARTpool: All four siRNAs are pooled together as a single reagent in one tube.
    • Set of Four: All four siRNAs are provided as individual duplexes in four individual tubes.

Consider a proprietary siRNA modification pattern for enhanced nuclease resistance

  • siSTABLE modifications prevent degradation from exo-and endonucleases. siSTABLE is recommended when the siRNA will be exposed to a biological environment rich in nucleases, such as animal serum.
  • Accell modifications incorporate delivery-enhancing properties in addition to stability enhancement. Accell is recommended when target cell or tissue types are not amenable to standard modes of delivery.
  • See a list of recent publications using siSTABLE and Accell modified siRNA for in vivo experimentation.
     
  • ORDER custom siRNA with siSTABLE or Accell »

     

Carefully calculate the total siRNA quantity required

  • Consider the number of animals in each cohort, the number of treatments or doses that will be required, and the amount of siRNA required for each dose
  • In vivo experiments typically require a large quantity of siRNA. Synthesis capabilities can accommodate up to 10 grams by request, or up to 100 mg available online
  • Need help converting nmol to mg? Check our siRNA yield table.

Consider in vivo processing of your siRNA when toxicity to the animal is of concern

  • A specialized post-synthesis siRNA processing procedure including counter-ion (Na+) exchange, sterile filtration, desalting, and endotoxin testing
  • Available with or without HPLC purification. For more information, review our siRNA purification and processing options.
  • For more information on important considerations for in vivo experimentation see our Technical Note: "In vivo RNAi: Biodistribution, Delivery, and Applications"
 

ON-TARGET modification ensures antisense strand bias


ON-TARGET Modification antisense strand bias

The ON-TARGET modification promotes correct strand uptake by blocking the sense (passenger) strand from being taken up by the RISC process. This is a useful method for ensuring antisense (guide) strand processing.

All proprietary siRNA modifications (ON-TARGETplus, Accell, siSTABLE) incorporate this sense-strand modification to promote antisense (guide) strand-facilitated silencing.

ON-TARGETplus siRNA dual-strand modification pattern for reduction of off-targets


ON-TARGET Plus dual-strand modification

A 2006 publication demonstrates that off-target effects are primarily driven by antisense strand seed activity.1 Therefore, sense strand inactivation alone does not decrease the total number of off-target genes. ON-TARGETplus modifications account for both strands:

  • Sense strand is modified to prevent interaction with RISC and favor antisense strand uptake
  • Antisense strand seed region is modified to minimize seed-related off-targeting

The ON-TARGETplus modification pattern dramatically reduces off-targets. Off-target effects induced by the indicated siRNAs were quantified using microarray analysis. For each target, three different siRNAs were used: unmodified, sense strand-inactivated, and ON TARGETplus-modified. Data shown represents genes down-regulated by two-fold or more. HEK293 cells were transfected with 100 nM siRNA using 0.2 µL of DharmaFECT 1. Data was analyzed at 24 hours.

1 Jackson, A.L. et al. "Position-specific Chemical Modification Increases Specificity of siRNA-mediated Gene Silencing." RNA 12.7 (2006) 1197-1205.

Accell delivery and gene silencing in cardiomyocytes


Accell delivery and gene silencing cardiomyocytes

Neonatal rat ventricular myocytes were incubated with 1 µM Accell Green (A; Cat# D-001950-01) or Red (B; Cat# D-001960-01) Non-targeting siRNA for 72 hours in Accell delivery media (Cat# B-005000). Nuclei were stained with DAPI (blue). Labeled control uptake showed diffuse cytoplasmic localization in nearly all cells.

The bar graph indicates the level of gene silencing achieved with Accell GAPD Control siRNA (Cat# D-001930-03) and Pool (Cat# D-001930-30) control reagents when used with neonatal rat ventricular myocyte (NRVM) media or Accell delivery media.

Myocytes were prepared as described in Maass AH & Buvoli M. Cardiomyocyte preparation, culture, and gene transfer. Methods Mol Biol. 2007;366: 321-30. mRNA expression was determined by QuantiGene branched DNA assay (Panomics).

siSTABLE-modified siRNAs resist degradation by nucleases


siSTABLE modified siRNAs resist degradation by nucleases

Conventional siRNA is degraded within minutes in serum-containing environments, making in vivo use of siRNA problematic. This graph presents evidence that the siSTABLE modification pattern dramatically extends the half-life of siRNA in the presence of 100% human serum as compared to Stealth RNAi (Invitrogen).

Accell siRNA also includes these stability-enhancing modifications, as well as providing delivery to difficult-to-transfect cells without a transfection reagent.

5 Fluorescein

5'-Fluorescein

Description: Fluorescein is often used in fluorescence experiments to demonstrate the kinetics of folding or substrate binding. Fluorescein is also used as a donor to track optimal changes related to folding or substrate binding to intermolecular interactions.

  • Mol. Wt.: 537.46
  • Special Note: Light sensitive. Fluorescein is very sensitive to photo bleaching.
  • Absorbance/Fluorescence: 494/520 nm

Reference: Science 266: 785-789 (1994), EMBO J. 17: 2378-2391 (1998)

5 DY547 CY3 Alternate

5'-DY547 (Cy3™ alternate)

For more information please contact Technical Support:

ts.dharmacon@ge.com
1-800-235-9880 (US)
00800-222 00 888
+44 (0) 845 3 63 04 25 (UK / EMEA)

  • Mol. Wt.: 491.25 g/mol
 
3'-DY547

3'-DY547 (Cy3™ alternate)

For more information please contact Technical Support:

ts.dharmacon@ge.com
1-800-235-9880 (US)
00800-222 00 888
+44 (0) 845 3 63 04 25 (UK / EMEA)

  • Mol. Wt.: 563.30 g/mol
 
5 Tamra-hexyl Linker

5'-TAMRA-hexyl linker

Description: TAMRA is a strongly absorbing dye with a wide variety of applications. This modification is coupled from the 5'- or 3'-end of an oligonucleotide to either the 5th or 6th position of the dye.

  • Special Note: Light sensitive
  • Absorbance/Fluorescence: 565/580 nm

References: Nucl. Acids. Res. 24: 4535-4542 (1996), Biochem. 39: 14487-14484 (2000)

5 DY647 CY5 Alternate

5'-DY647 (Cy5™ alternate)

For more information please contact Technical Support:

ts.dharmacon@ge.com
1-800-235-9880 (US)
00800-222 00 888
+44 (0) 845 3 63 04 25 (UK / EMEA)

  • Mol Wt.: 517.26 g/mol
 
5 Dy677 CY5.5 Alternate

5'-DY677 (Cy5.5™ alternate)

For more information please contact Technical Support:

ts.dharmacon@ge.com
1-800-235-9880 (US)
00800-222 00 888
+44 (0) 845 3 63 04 25 (UK / EMEA)

  • Mol Wt.: 617.29 g/mol
 

Modification Name: 5’-Cy3

Modification Code: Cy3

Unit Molecular Weight: 507.59 g/mol

Cy3 Extinction Coefficient: 136,000

Excitation/Emission Max: 547 nm/563 nm

Unit Structure:

Unit Structure: 5’-Cy3

Modification Name: 3’-Cy3

Modification Code: Cy3-3’

Unit Molecular Weight: 800.85 g/mol

Cy3 Extinction Coefficient: 136,000

Excitation/Emission Max: 547 nm/563 nm

Unit Structure:

Unit Structure: 3’-Cy3  

Modification Name: 5’-Cy5

Modification Code: Cy5

Unit Molecular Weight: 533.63 g/mol

Cy5 Extinction Coefficient: 250,000

Excitation/Emission Max: 646 nm/662 nm

Unit Structure:

Unit Structure: 5’-Cy5

Modification Name: 3’-Cy5

Modification Code: Cy5-3’

Unit Molecular Weight: 826.88 g/mol

Cy5 Extinction Coefficient: 250,000

Excitation/Emission Max: 646 nm/662 nm

Unit Structure:

Unit Structure: 3’-Cy5  

Modification Name: 5’-Cy5.5

Modification Code: Cy5^5

Unit Molecular Weight: 633.75 g/mol

Cy5.5 Extinction Coefficient: 209,000

Excitation/Emission Max: 688 nm/707 nm

Unit Structure:

Unit Structure: 5’-Cy5.5

Modification Name: 3’-Cy5.5

Modification Code: Cy5^5-3’

Unit Molecular Weight: 927.00 g/mol

Cy5.5 Extinction Coefficient: 209,000

Excitation/Emission Max: 688 nm/707 nm

Unit Structure:

Unit Structure: 3’-Cy5.5  

Modification Name: 5’-Cy5

Modification Code: Cy5

Unit Molecular Weight: 533.63 g/mol

Cy5 Extinction Coefficient: 250,000

Excitation/Emission Max: 646 nm/662 nm

Unit Structure:

Unit Structure: 5’-Cy5  

Modification Name: 5’-Cy3

Modification Code: Cy3

Unit Molecular Weight: 507.59 g/mol

Cy3 Extinction Coefficient: 136,000

Excitation/Emission Max: 547 nm/563 nm

Unit Structure:

Unit Structure: 5’-Cy3  

Modification Name: 5’-Cy5.5

Modification Code: Cy5^5

Unit Molecular Weight: 633.75 g/mol

Cy5.5 Extinction Coefficient: 209,000

Excitation/Emission Max: 688 nm/707 nm

Unit Structure:

Unit Structure: 5’-Cy5.5

Modification Name: 3’-Cy5

Modification Code: Cy5-3’

Unit Molecular Weight: 826.88 g/mol

Cy5 Extinction Coefficient: 250,000

Excitation/Emission Max: 646 nm/662 nm

Unit Structure:

Unit Structure: 3’-Cy5  

Modification Name: 3’-Cy3

Modification Code: Cy3-3’

Unit Molecular Weight: 800.85 g/mol

Cy3 Extinction Coefficient: 136,000

Excitation/Emission Max: 547 nm/563 nm

Unit Structure:

Unit Structure: 3’-Cy3  

Modification Name: 3’-Cy5.5

Modification Code: Cy5^5-3’

Unit Molecular Weight: 927.00 g/mol

Cy5.5 Extinction Coefficient: 209,000

Excitation/Emission Max: 688 nm/707 nm

Unit Structure:

Unit Structure: 3’-Cy5.5  

What is an NHS Ester?

An NHS ester is a reactive group found on the dye that provides the functionality for labeling amino groups found on the oligonucleotide.

Some DyLight dyes are available as phosphoramidites (our standard dyes), and others as NHS esters (our non-standard Alexa alternatives). If applicable, we recommend DyLight dyes as a first choice since this form is most compatible with our 2'-ACE RNA synthesis chemistry and will therefore be more likely to produce higher yields.

siRNA Yield Table


For unmodified siRNA, the following approximate yields can be expected:
  Standard (A4) Purified in vivo in vivo HPLC
nmol mg nmol mg nmol mg nmol mg
0.025 µmol scale 20 0.25 - - - - - -
0.05 µmol scale 40 0.5 20 0.25 25 .3 - -
0.2 µmol scale 150 2 80 1 100 1.3 50 0.65
0.4 µmol scale 300 4 160 2 200 2.6 100 1.3
1.0 µmol scale 750 10 320 4 500 6.6 250 3.3
2.0 µmol scale 1500 20 750 10 1000 13 500 6.6
5.0 µmol scale 3750 50 1875 25 2500 33 1250 16
10.0 µmol scale 7500 100 3750 50 5000 66 2500 33
Pre-designed siRNAs are available in the following formats:
  • Individual duplexes: Four individual siRNAs are pre-designed for every gene in human, mouse and rat. Order 1, 2, 3 or 4.
  • SMARTpool: All four siRNAs are pooled together as a single reagent in one tube.
  • Set of Four: All four siRNAs are provided as individual duplexes in four individual tubes.