siRNA design needs to be completed by software, but there are some general rules:
Note: The choice of RNAi has certain randomness for any target gene.
siRNA works at the mRNA level rather than the protein level. In order to design siRNA, a precise target mRNA nucleotide sequence is required. Due to the degenerate nature of the genetic code, it is impossible to accurately predict the correct nucleotide sequence from the peptide sequence.
Since the function of siRNA is to cut the mRNA sequence, it is very important to use mRNA nucleotide sequence instead of genomic sequence for siRNA design. mRNA pretreatment removes intron sequences contained in the genome sequence. Designs using genomic information may inadvertently target introns, so siRNA cannot silence the corresponding mRNA.
We recommend using 1X TE buffer (10 mM Tris-HCl, pH 7.5; 0.1 mM EDTA; no RNase) to dissolve single-stranded RNA. This solution can buffer pH and chelate metal ions, and then reduce RNA degradation. You can also use RNase-free water to dissolve lyophilized dsRNA (10 mM Tris-HCL, pH 8.0, 20 mM NaCl, 1 mM EDTA). We recommend a stock concentration between 20-100 uM, depending on the supply of the initial quantity of siRNA. For example, resuspending 5 nmol in 250 uL of the buffer will produce a 20 uM stock solution.
siRNAs are shipped as a dry pellet, should be resuspended in an RNAse-free solution before use, and stored in a refrigerator at -20 ° C or -80 ° C. We recommend resuspending siRNA to the appropriate stock concentration (20-100 uM) and storing it in small aliquots to avoid multiple freeze-thaw cycles. siRNA should not undergo more than 5 freeze-thaw cycles, and can usually be kept stable for more than 6 months. If degradation needs to be considered, the integrity of the siRNA duplex can be evaluated on the analytical PAGE gel.
It is recommended that each aliquot of siRNA or RNA does not exceed 4-5 freeze-thaw cycles to ensure product integrity.
The duplex of RNA is much more resistant to nucleases than the single strand. However, it is always recommended to maintain sterile and RNAse-free conditions as a precautionary measure. The dried siRNA powder is stable at room temperature for 2-4 weeks but should be placed at -20 ° C or -80 ° C for long-term storage (under this condition, the dried siRNA will be stable for at least one year).
The non-targeted siRNA control design is very important to distinguish sequence-specific silencing from non-specific effects in RNAi experiments. Because non-specific effects may be siRNA concentration-dependent or caused by transfection reagents or other stimuli, it is important to use negative control siRNA at the same concentration as gene-specific siRNA.
The effective delivery of siRNA is essential for achieving significant gene silencing. Positive control siRNA can be used to optimize delivery conditions and monitor transfection efficiency throughout the experiment. The widely validated siRNA targeting the housekeeping gene is very suitable as a positive control.
We provide a variety of positive and negative controls in the siRNA control product line, as well as fluorescent controls for siRNA experiments. For more information, please contact BOC RNA technical support email@example.com.
Non-targeting siRNA controls are important to help determine that any decrease in gene expression levels observed using gene-specific siRNAs is related to sequence-specific RNAi events. The event of down-regulation of gene expression may be due to the stress response of the cell to a certain transfection reagent or technology. Without a negative control, researchers may mistakenly interpret this extensive non-specific silencing as true gene-specific silencing.
In order to control non-RNAi related or non-specific effects, BOC RNA provides a variety of RNAi experimental products. Please refer to our siRNA control for more information.
The stock concentration is calculated as follows: (amount of siRNA, nmol) / (resuspended volume, uL) = stock concentration, umol/L.
Example: You have purchased 20 nmol of siRNA and want to use a 50 uM stock solution.
Equation: a. (20nmol)/? uL = 50 umol/L b. Solve the unknown: uL = (20 nmol) (1 L/50 umol) c. Unit conversion: uL = (20 nmol) (1 L/50 umol) (1 umol/1000 nmol) (10exp6 uL/1 L) d. Answer: uL = 400 uL Therefore, use 400 uL of 1X RNase-free buffer to resuspend 20 nmol of siRNA to make a 50 uM solution.
There is a precise formula for the conversion between OD and nmol of RNA oligonucleotides, but in general, for 21bp siRNA oligonucleotides, there is a simple calculation method as follows: 1 OD duplex = 3 nmol = 40ug.
The molecular weight of siRNA is switched between nmol and ug. If the specific molecular weight is unknown, you can use the average molecular weight of the siRNA, which is 13,300 g/mol. For example, if you have 5 nmol of siRNA, the conversion will be performed using the following steps: Equation: a. ug = (5 nmol) (13,300 g/mol) b. Unit conversion: ug = (5 nmol) (13,300 g/mol) (mol/10exp9 nmol) (10exp6 ug/g) c. Answer: ug = 66.5 ug, therefore, 5 nmol of siRNA is 66.5 ug.
siRNAs specifically designed to target human genes will not silence homologs from other species. So far, even with a high degree of identity between the gene sequences of two species, in siRNAs designed for genes in a particular-species, it is rarely possible to cross-target another species. In some cases, siRNAs can be specifically designed to function in two or more species, but this requires careful siRNA design and bioinformatics analysis. For more information, please contact BOC RNA technical support firstname.lastname@example.org.
|Abbreviation Name||Absorption Wavelength||Emission Wavelength|
|6-FAM 6-carboxy-fluorescein||494 nm||518 nm|
|TET 5-tetrachloro-fluorescein||521 nm||538 nm|
|HEX 5-hexachloro-fluorescei||535 nm||553 nm|
|TAMRA tetramethyl-6-carboxyrhodamine||560 nm||582 nm|
|ROX 6-carboxy-x-rhodamine||587 nm||607 nm|
|Cy3 Indodicarbocyanine||552 nm||570 nm|
|Cy5 Indodicarbocyanine||643 nm||667 nm|
|CY5.5 Indodicarbocyanine||684 nm||710 nm|
|Cy7 Indodicarbocyanine||743 nm||767nm|
|Dyomics681||691 nm||708 nm|
|Dyomics781||783 nm||800 nm|
|AMCA||350 nm||450 nm|
|JOE||520 nm||548 nm|
|BHQ-1||480 nm||580 nm|
|BHQ-2||599 nm||670 nm|
|Texas Red||589 nm||615 nm|
|Methlyene Blue||609 nm, 668 nm|
The uptake of labeled pre-synthesized siRNA can be easily detected by flow cytometry or fluorescence confocal microscopy. We recommend that you use protocols and procedures specific to the optical system and software package to detect fluorescently labeled cells.
When using siRNA in animal models, special consideration should be given to the stability of the siRNA and its toxicity to animals. Special chemical modification can significantly enhance the stability of siRNA in a nuclease-rich environment so that the siRNA reaches the target tissue to achieve gene silencing.
We provide customized chemically modified siRNAs services, all of which have undergone HPLC treatment including counter ion (Na) exchange, desalting, sterile filtration, and endotoxin testing.
Because there is relatively little accumulation of siRNA work in vivo, it is difficult to specify exactly how much siRNA is needed for these experiments. We recommend conducting a literature search (PubMed or HighWire) to identify other in vivo studies using similar routes of administration and target tissue to determine the amount of siRNA required.
We recommend using the final concentration of siRNA at a screening concentration of 100 nM * for initial experiments and then reducing the concentration to use alternative reagents while maintaining knockdown effects. Prepare 1 mL of 100 nM siRNA solution with 1 nmol siRNA. This volume will usually rupture and be transfected into a complete 96-well or 24-well plate.
* 100 nM = 100 nanomoles/liter = 100 pmol/mL = 100fmol/uL
The off-target effect is related to the siRNA itself; the most common reason is that the sense strand or antisense strand is partially complementary to the unintended target. These effects are concentration-dependent, so the best way to minimize these off-target effects is to reduce the siRNA concentration. Bioinformatics methods can be used to select sequences that are unlikely to exhibit a high degree of off-target. In addition, we recommend the use of chemical modifications to reduce the off-target effect of the two chains.