General siRNA FAQs

 

 

 

Not at this time

 

We use the Tuschl protocol. It has been validated internally. For annealing, we incubate 20 µM single-stranded 21-nt RNAs 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. The solution can be stored frozen at -20 °C and freeze-thawed many times. If a higher concentration of siRNA has been requested, we will use more than 20 µM of each strand.

 

In the vast majority of cases desalt (DST) siRNA duplexes will more than suffice to meet your needs. The process of purification basically enriches oligonucleotides for the full-length sequences. Our siRNAs are duplexed together from single-stranded synthetic RNA molecules that are individually "desalted" to remove by-products of the synthesis reactions, quality controlled, and quantified. 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. The duplex itself is then separately run on a non-denaturing PAGE gel to check for on its overall integrity.

Contact technical service if you have additional questions.

RNA interference (RNAi), also known as post-transcriptional gene silencing, is a natural biological mechanism in which small interfering RNA (siRNA) duplexes induce potent inhibition of gene expression by destroying messenger RNA (mRNA). These siRNA duplexes are produced naturally when an enzyme, Dicer, cleaves long double-stranded RNA (dsRNA). Alternatively, synthetic versions of siRNA can be introduced into the cell, thus triggering the remainder of the RNAi pathway. These 21-23 nucleotide fragments, termed siRNA, associate with an RNase containing complex to form the RNA-induced silencing complex (RISC). ATP-dependent unwinding of the siRNA duplex is required for activation of RISC. This complex unwinds the siRNA duplex and releases the sense strand. The RISC-bound antisense strand then serves as a guide for targeting the activated complex to complementary mRNA sequences,* resulting in subsequent mRNA cleavage and degradation. In effect, only catalytic amounts of siRNA are required for destruction of mRNA, resulting in the knockdown or silencing of the target gene and diminished protein expression.

 

*Hammond, S.M., et al., Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet., 2, 110-9 (2001).

The actual MW is listed on the Quality Assurance Document in g/mol. The average MW of a 21 bp duplex for siRNA is ~13,300 g/mol.

 

There are multiple reasons why this may be the case:

1. The sample may not be homogeneously mixed.

2. Differences in instrumentation for quantifying siRNA may lead to differences in apparent values. Dual beam UV-VIS spectrophotometers are recommended.

3. The sample may be too concentrated. Absorbance values are most accurate between 0.15 and 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.

Our R&D performed an accelerated stability study on several siRNA duplexes. They found that dry siRNA duplexes kept at -20 °C for three years are 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 the two year mark. 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 six months at - 20 deg C in a non-frost-free freezer, we recommend that the solution is not freeze/thawed more than 3-5 times.

 

In the lyophilized form the duplexes stable for much longer at - 20 deg C.

Yes. Samples are shipped as dried pellets and are stable at room temperature for 2-4 weeks. Upon receipt, we recommend to store siRNAs at -20ºC or -70ºC to -80ºC.

 

Dried siRNAs are shipped at ambient temperature. Upon receipt, prior to reconstitution, store at –20 °C. For extended storage of reconstituted product, freeze at –20 °C in working aliquots. Repeated freezing and thawing or storage in “frost-free” freezers is not recommended.