Custom siRNA synthesis

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 modification ensures 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

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 without a transfection reagent

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

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.

*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   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)	494	520		-	both*
TAMRA   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)	565	580		-	both*
Cy3   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: 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:	547	563		-	both*
Cy5   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: 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:	646	662		-	both*
Cy5.5   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: 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:	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 Scientific 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.

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 Scientific Support for information regarding recommendations for purification or purity estimates for unpurified material.

siRNA Processing Options:

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

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.
   
• dye

  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"