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MISSION® siRNA FAQs

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.

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

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.

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 some sequence-specific stability differences--only some sequences showed any signs of degradation at two years. This is only representative of duplexed siRNA.

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.

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.

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.

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.

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.

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

Yes, we offer Custom siRNA with several available.

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.

Our modifications page for additional details on the above options.

Yes, you can order this a Custom siRNA.

No

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.

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.

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

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.

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, refer 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.

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.

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 (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

We recommend a concentration of 50 – 100 µM.

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), (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.

Refer to this article.

Use the following formula:

nmol to ug formula

siRNA is dsRNA:

  • 1 A260 unit = 60 µg dsRNA

Therefore

  •  1 OD siRNA = 5 nmol = 60 µg

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

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.

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.

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.

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.

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

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

Both transfection reagents have a technical bulletin with protocols on the product ordering pages.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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. 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.

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.

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.

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.

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

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