siRNA FAQ

If you do not find the answer here in our FAQ (frequently asked questions), please send a request here.

General

Duplex Design

Experiment & Analysis

in vivo Applications & N-TER™ Use

Validation & Guarantee

What is siRNA?

siRNA (short [or small] interfering RNA) is a class of small double-stranded RNA molecules that silence gene expression by inducing the RISC (RNA-induced silencing complex) to cleave mRNA.

What is the average molecular weight of siRNA?

For a typical 21mer duplex, it is approximately 13,300. The actual molecular weight is listed on the Standard Certificate of Analysis / Technical Datasheet.

What is the recommended storage condition for siRNA?

For short-term storage, single-stranded RNA oligonucleotides should be stored in TE buffer at -80°C. For the long-term, storage at -80°C as an ethanol precipitate is the best option. Taking precautions to minimize exposure to RNases will increase shelf life.

If a -80°C freezer is unavailable, storage at -20°C will likely suffice. If resuspended, store as working aliquots. Repeated freezing and thawing or storage in “frost-free” freezers is not recommended.

How long are siRNA stable?

Our R&D team performed an accelerated stability study on several siRNA duplexes. Dry siRNA duplexes kept at -20°C for three years were stable. Dry siRNA duplexes kept at room temperature only showed signs of minor degradation after two years. Our study did see some sequence-specific stability differences--only some sequences showed any signs of degradation at two years. This is only representative of duplexed siRNA.

How many times can I freeze and thaw reconstituted siRNAs?

Repeated freezing and thawing or storage in “frost-free” freezers is not recommended. siRNA may be stored for up to 6 months at -20°C. In a non-frost-free freezer, we recommend that the solution is not freeze or thawed more than 3–5 times. In the lyophilized form, the duplexes are stable for much longer at -20°C.

What level of purification do I need for my siRNA?

In most cases, Desalt will be more than enough to meet needs. The duplexing of the separate strands self-selects for the full-length oligonucleotides, as this process relies upon a perfect match between the self-complementary strands.
Depending on manufacturing site, the duplex itself may then be separately run on a non-denaturing PAGE gel to check its overall integrity.

How do you anneal siRNA simplexes to form a duplex?

We use the Tuschl protocol, which we have validated. For annealing, we incubate 20 µM single-stranded 21-base RNA in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90°C, followed by 1 hour at 37°C. If a higher concentration of siRNA is needed, then we will use more than 20 µM of each strand.

Why does my calculated amount of siRNA in solution differ from that on the Standard Certificate of Analysis / Technical Datasheet

There are several possible reasons:

  1. The sample may not be homogeneous.
  2. Differences in instrumentation may lead to differences in apparent values. We recommend Dual beam UV-Vis spectrophotometers.
  3. The sample may be too concentrated. Absorbance values are most accurate between 0.15 – 0.6 and within the linear range of a standard curve.
  4. The sample may be too diluted. Measurements with dilutions of small volumes (1 – 1.5 μL) are more susceptible to variation.

We received siRNA 2 weeks ago, and it is sitting on my benchtop. Is it OK?

Yes, samples are shipped dry and are stable at room temperature for 2–4 weeks. Upon receipt, we recommend to store siRNA at –20°C, but -80°C is preferable.

Can you make siRNA with 5' or 3' tags?

Yes, we offer Custom siRNA with several available 5' or 3' modifications.

Can you make siRNA with modified bases?

Yes, we offer Custom siRNA with several available.

Could you please provide the function of modified bases and 5' or 3' tags?

Common choices include:

  • Amino-Modifier C6, Amino-Modifier C7: These modifications are used to either block one or both ends of the simplexes (using an amine on the 5’ end of the sense strand facilitates the desirable loading of the antisense strand into RISC) or to attach an NHS-ester dye which may not be available as a phosphoramidite (direct addition to one of the simplexes during synthesis) or perhaps is not compatible with the chemistry of cleaving and deprotection of the RNA.
  •  Biotin: This is typically used to attach a Streptavidin-tagged molecule, or to be used to separate the siRNA from other molecules.
  • Thiol-Modifier C6 S-S: Similar to amines, it can be used to attached other molecules via a disulfide bond.
  • Cyanine 3, 5, & 5.5: Used for detection of one of the simplexes via fluorescence, which aids visualization and / or determination of transfection efficiency.
  • 6-FAM™: Used for detection of one of the simplexes via fluorescence, which aids visualization and / or determination of transfection efficiency.
  • Phosphate: May aid in the strand selection process similar to amine modifications.
  • 2'-OMe RNA: Resists cellular nuclease degradation and offers increased specificity.
  • Phosphorothioate: Resists cellular nuclease degradation.
  • Cholesterol: Improved permeation through the plasma membrane.

See our modifications page for additional details on the above options.

Can you make the siRNA I design?

Yes, you can order this a Custom siRNA.

Do you offer GMP siRNA?

No

What overhangs do you use on your predesigned siRNA, and why are those chosen?

We use dTdT overhangs. According to *Elbashir et al. 2001, "The thymidine overhang was chosen because it may enhance nuclease resistance of siRNAs in the cell culture medium and within transfected cells."

Early in RNAi research, leaders in the field chose dTdT overhangs, so to some extent, it is a historic choice. siRNA are often designed with dTdT overhangs, but UU or UG overhangs are also common. Several Elbashir et al. papers from 2001 outline a set of rules for siRNA design based on a limited number of genes and systems, but researchers continue to use dTdT and UU overhangs.

*Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K & Tuschl T (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature, 411, 494-498.

How does the Rosetta algorithm work?

The Rosetta siRNA Design Algorithm utilizes Position-Specific Scoring Matrices (PSSM) and knowledge of the all-important siRNA seed region to predict the most effective and specific siRNA sequences for the target gene of interest. Additionally, the Rosetta siRNA Design Algorithm has been trained with feedback from over 3 years of gene-silencing experiments, which ensures that the algorithm’s in-silico rules are guided and bolstered by real-world empirical evidence.

Why do you list siRNA from several different NM sequences? Should I choose the #1 of each of the NM sequences listed, or should I choose numbers 1, 2, and 3 from one NM sequence?

When there is more than one NM sequence listed, this means that there are alternate transcripts for that gene. Our Rosetta Algorithm is run separately for each cDNA transcript. Choose 3 siRNA from one RefSeq to ensure 3 unique target sequences.

How do I know which predesigned siRNA I should I order? What does the numbering mean?

Critical rules incorporated into the Rosetta algorithm lead to siRNA of increased target specificity and knockdown for low abundance messages. The ranking for predesigned siRNA is from 1 to 10, with 1 being the highest rank and 10 being the lowest. Positive control siRNA or validated siRNA are called out via the "siRNA Type column."

Can siRNA integrate into the genome so that I can get stable knocked down cell lines?

No, if you need stable knocked down cell lines, please see our shRNA product line.

How long does siRNA silence a gene?

Gene silencing resulting from siRNA can be assessed as early as 24 hours post-transfection. The effect most often will last from 5–7 days. However, the duration and level of knockdown are dependent on the cell type and concentration of siRNA. Transfections may be repeated to maintain silencing.

How can I enhance the effect of my siRNA?

It has been demonstrated that adding enoxacin to siRNA experiments caused an increase in the ability of the siRNA to silence the target gene. For more information, see this paper:

Shan G, Li Y, Zhang J, Li W, Szulwach KE, Duan R, Faghihi MA, Khalil AM, Lu L, Paroo Z, Chan AW, Shi Z, Liu Q, Wahlestedt C, He C & Jin P (2008). A small molecule enhances RNA interference and promotes microRNA processing. Nat Biotechnol, 26, 933-940.

Another reference which may be helpful is:

Duxbury MS, Ashley SW & Whang EE. (2005). RNA interference: A mammalian SID-1 homologue enhances siRNA uptake and gene silencing efficacy in human cells. Biochem Biophys Res Commun, 33, 459-63.

Do you offer pooled siRNA?

Yes, custom or predesigned siRNA can be pooled with a popular format consisting of 4 duplexes at 5 nmol each combined into one tube (20 nmol pooled)) plus the exact same 4 duplexes also at 5 nmol each in separate tubes (another 20 nmol individual). However, our sophisticated liquid handlers allow for a wide range of other possibilities.

In addition, another product option is esiRNA, which consists of siRNA resulting from cleavage of long dsRNA (double-stranded RNA) with E. coli RNase III. Since esiRNA are pools of siRNA that all target the same mRNA, they are highly specific.

Do you have a recommended procedure for pooling individually ordered duplexes?

Yes, for each duplex that will be pooled, take half of its quantity and following these steps:

  1. Resuspend each duplex with 100 µL of molecular biology grade water (Material# W4502). There is no need for buffer as it is already included in the dry siRNA material
  2.  Remove 50 µL from each of the individual siRNA and transfer to a new tube.
  3. The remaining siRNA solution in each tube can be used for additional studies

At what concentration should I make my siRNA stock solution?

We recommend a concentration of 50 – 100 µM.

Can I resuspend the dried siRNA in RNase-free water instead of 1X TE?

We do not recommend resuspending dried siRNAs in 1X TE.

Before resuspending siRNA, briefly centrifuge the tubes. siRNA is dried down in the presence of buffer (show below). Therefore, the siRNA should be resuspended in molecular biology grade water (DNase- and RNase-free), Material# W4502.

siRNA Buffer:

  •  Potassium Acetate (100 mM)
  • HEPES (30 mM)
  • Magnesium Acetate (2 mM)

Note: siRNA are susceptible to degradation by nucleases introduced during handling. Use RNase-free reagents, tubes, and filtered/barrier pipette tips. Always wear gloves when handling siRNA.

How do I calculate the concentration of the siRNA in my sample?

See this article.

How do I convert nmol to ug?

Use the following formula:

How does OD relate to nmol and µg?

siRNA is dsRNA:

  • 1 A260 unit = 60 µg dsRNA

Therefore

  •  1 OD siRNA = 5 nmol = 60 µg

How much siRNA do I use for various plate/well sizes? How many wells of a 6 well plate can I transfect with 2 OD (10 nmol) siRNA?

Please see the following table (Note: final siRNA concentration = 30nM; 1 nmol = 1000 pmol):

Typical siRNA experiment reagent amounts
 

Format Transfection Reagent (µL) siRNA (pmol) cells / well Final Volume (mL)
96-Well 0.3 – 1 3 6,000 0.1
24-Well 1 – 3 15 40,000 0.5
12-Well 2 – 4 30 80,000 1.0
6-Well 3 – 6 75 200,000 2.5

You can transfect approximately 10,000/75 = 130 wells

What siRNA concentration(s) should be used when starting a new experiment?

We recommend testing siRNA in small pilot experiments to validate the best concentration for every cell type and new experimental procedure, using a concentration range from 5 – 100 nM in culture medium. We find that 30 nM is typically a reasonable starting concentration. The lowest concentration that results in the desired knockdown should be used. This will help reduce potential off-target effects.

If I am overexpressing a gene in my cells, will siRNA be able effectively silence it?

Overexpressed genes have been shown to be successfully silenced using siRNA. It is difficult to predict by how much the overexpression will be reduced. It may be necessary to balance final siRNA concentration with any toxicity/cellular innate immunity issues to see a functional knockdown.

The gene I want to silence is very stable. How will this affect my siRNA experiment?

Even stable genes, such as the common reference genes GAPDH and Cyclophilin B, are effectively silenced using typical siRNA approaches. While performing cell viability analysis is always a good control, this becomes more important when pursuing the silencing of potentially essential genes that may be represented by those most stably expressed.

Our predesigned siRNA has been shown to be effective at silencing high-expression, stable mRNA. While the protein of the gene may be quite stable, the stability of the mRNA should not be a huge factor when using siRNA. RNAi directly targets mRNA for cleavage and subsequent degradation by cellular nucleases. Always attempt to optimize the correct amount of siRNA to your mRNA of interest for proper empirical determination.

What is the best approach to elucidate individual siRNA from a heterogeneous (pooled) population?

If you want pooled siRNA, rather than pooling the individual siRNA yourself, the best method would be to order the siRNA contained in the pool as individuals and test the individuals in your experiment. We sell individual siRNA at concentrations appropriate for pooling and testing.

How do I transfect suspension cells?

We do not offer a transfection reagent appropriate for suspension cells. There are several effective reagents available from other manufacturers.

Do you have a protocol and/or recommended reagents for siRNA transfection?

There is no one single protocol for transfection. The protocol depends on the transfection reagent you choose to use. We offer two three such reagents:

All three transfection reagents have technical bulletin with protocols on the product ordering pages.

How do I optimize transfection conditions for my cell line?

To identify optimal transfection conditions for a cell line, we recommend setting up a matrix of cell density and siRNA concentration. It is a good idea to use a validated and highly effective siRNA for optimization. A matrix of serum versus no serum should also be tested if appropriate for the cell line. Regardless of the cell type, the N-TER™/siRNA complexes will enter the cell quickly, so there is no advantage to incubating the cells longer. Most adherent cell lines can be transfected with N-TER™ regardless of whether they are immortalized or primary.

What cells should I use for siRNA experiments?

Cell lines are often selected as a function of the desired investigation. For example, investigations focusing on liver function will often use HepG2 or comparable cell lines since these lines are derived from liver cells.

Which cell lines have been tested with your siRNA?

Most of our studies have been conducted in the HeLa and A549 cell lines as these two cell lines are commonly used in life science research.

What siRNA controls do you recommend?

For positive controls, we offer Positive Control siRNA. For negative controls, we recommend MISSION® siRNA Universal Negative Controls.

We can also synthesize custom positive or negative controls. Many researchers have their own scrambled sequence that they prefer to use, and some use luciferase or eGFP siRNA controls. These can be ordered the same as our Custom siRNA.

The advantage with the luciferase (or other marker genes) is you can use these siRNA also as positive controls when co-transfected with the respective marker gene.

I am seeing an off-target effect. How can I minimize it?

It can be difficult to obtain high-quality data from RNAi experiments because of off-target differential gene expression. There is no clear answer on how to solve the problem. We offer esiRNA for knockdown experiments. This product leads to effective knockdowns while reducing off-target effects. esiRNA achieves this through specific designs and the ability to use lower concentrations.

What is a typical off-target effect of siRNA?

Since siRNA recognizes and binds to the 3’ UTR in genes, it has the potential to behave similarly to miRNA (microRNA), which can result in unintended off-target silencing. On average, one third of mammalian mRNA are targets of one or more miRNA. Therefore, if siRNA enters the miRNA pathway, this may elicit hundreds of off-target silencing effects.

Do you have any data for in vivo quality siRNA?

Yes, we teamed up with PerkinElmer (Bioo Scientific™) to validate our MISSION® in-vivo-quality siRNA with Bioo Scientific™'s MaxSuppressor™ In Vivo RNA-LANCEr II in vivo delivery agent. Consult PubMed to identify similar studies to determine the specific delivery needs.

Do you have siRNA that is specifically for in vivo experiments?

Yes, we offer MISSION® in-vivo-quality siRNA. The product is available in iScale (intermediate scale) quantities (mg and g) for animal and larger-scale studies.

How long does siRNA-mediated knockdown last in vivo?

See the references on the landing page for MISSION® in-vivo-quality siRNA. In addition, consult PubMed to identify similar studies to determine the specific delivery needs.

How much siRNA do I need for in vivo experiments in mice?

This can depend on the delivery reagent you choose. Refer to the manufacturer’s technical bulletin for the protocol.

Is siRNA toxic in vivo?

See the references on the landing page for MISSION® in-vivo-quality siRNA. In addition, consult PubMed to identify similar studies to determine the specific delivery needs.

What factors should I address before initiating an in vivo study?

You need to outline your experimental setup for in vivo studies. This includes the choice of model organism, route of delivery, whether to use a delivery reagent, dosage, and frequency of dosing. The concentration delivered will depend largely on the nature of the target, the expression pattern, and the size of the study.

What tissues have siRNA been delivered into?

See the references on the landing page for MISSION® in-vivo-quality siRNA. In addition, consult PubMed to identify similar studies to determine the specific delivery needs.

What controls should I use with an in vivo siRNA experiment?

See the references on the landing page for MISSION® in-vivo-quality siRNA. In addition, consult PubMed to identify similar studies to determine the specific delivery needs.

What delivery reagent should I use for siRNA in vivo?

See the references on the landing page for MISSION® in-vivo-quality siRNA. In addition, consult PubMed to identify similar studies to determine the specific delivery needs.

Do you have an easy way to do reverse transfections of siRNA with N-TER™?

Yes, click here to download our protocol.

Can I use N-TER™ to transfect my plasmid DNA?

No, N-TER™ will not work with plasmid DNA.

Can I use N-TER™ to transfect siRNA into HUVEC cells?

Yes, click here to download our protocol.

Can I use N-TER™ alone as a negative control?

No, use of the N-TER™ peptide alone may result in cellular cytoxicity. We recommend that you use a buffer-only control and a non-targeting siRNA for negative controls.

Can you compare N-TER™ to lipid-based transfection reagents?

Use of N-TER™ results in a higher assessed knockdown. In a comparison, 25 gene targets were examined, and 24 were silenced more when N-TER™ was used vs. a competitor's lipid-based transfection reagent. Viability was comparable where 7 of 25 targets demonstrated higher viability when a competitor's lipid-based transfection reagent was used. The mean overall difference in viability was only 7.1%. Average increase in knockdown was 22% using N-TER™ vs. a competitor's lipid-based transfection reagent at 5 µL. When a competitor's lipid-based transfection reagent was used at a higher concentration, viability was impacted as 19 of 25 targets showed decreased viability by an average of 13% when compared to N-TER™-treated cells. However, even when increased amounts of a competitor's lipid-based transfection reagent were used, silencing continued to be greater using N-TER™ in 18 of 25 instances.

N-TER™ is a peptide-based transfection system. Lipid-based reagents are typically targeted to endosomal compartments which can result in the production of inflammatory cytokines and global changes in gene expression (an off-target effect). Additionally, targeting of siRNA to the endosome can result in siRNA degradation. N-TER™ bypasses endosomal compartments resulting in fewer off-target effects and less degradation of the siRNA.

Does N-TER™ target siRNA to the nucleus or the cytoplasm?

N-TER™ has been modified so that it releases siRNA into the cytoplasm. Cytoplasmic release of siRNA has been shown to correlate with increased knockdown efficiency.

Does N-TER™ bind the siRNA through a covalent reaction?

No, N-TER™ associates with the siRNA in a non-covalent manner. While covalent attachment can increase the uptake of siRNA, release of the covalently attached siRNA can be a barrier for efficient and rapid knockdown. Non-covalently attached siRNA is released very rapidly into the cytoplasm of transfected cells resulting in rapid mRNA knockdown.

How do I scale-up my transfection reaction?

N-TER™/siRNA complex formation is less efficient in large volumes. We recommend that you scale up in 3X volumes. These 3X volumes can be combined after complex formation has taken place.

Do you have any references for N-TER™?

Yes, see selection citations here.

How do you validate your predesigned siRNA?

There are two parts to validation:

  • All predesigned siRNA are validated in silico using the Rosetta algorithm.
  • A subset of predesigned siRNA functionally validated as outlined.
       
    • Transfection reagents: Various transfection reagents were used to functionally validate the siRNA constructs.
    • siRNA concentrations: Various concentrations were used. The most common was 50 nM, and it was used for >60% of the validation work. 50 nM was used primarily because it was the concentration used to validate the Rosetta algorithm. Subsequently, we confirmed performance at much lower concentrations.
    • Cell lines: Nearly all validation testing was completed in HeLa cells.
    • Formats: 96-well plates.
    • Replication: We conducted at least three biological replicates and three technical replicates for each siRNA validated. Additionally, viability testing was done on nearly all validation experiments.
    • Times to harvest mRNA: Most measurements were obtained at 24 hours post-transfection.
    • Detection of knockdown: Branched DNA technology or qPCR.

Do you guarantee that your siRNA will knock down my gene?

We guarantee that two out of three of our predesigned siRNA per target gene will achieve knockdown efficiencies ≥75%. Custom designed siRNA are not guaranteed. Please see our full guarantee.  

How should I choose the 3 siRNA so that I have the benefit of your guarantee?

When starting a new experiment, we recommend choosing the top three predesigned siRNA.