siRNA Duplex

(1) The product is shipped in the form of lyophilized powder, the dry powder is attached to the wall of the tube and is easily dispersed when opened, so please centrifuge the tube for a few seconds before dilution to make the product gather at the bottom of the tube and open the cap carefully, cover the tube after dissolution, vortex and shake to mix to make full dissolution. It is recommended to store at -20°C or below, avoid repeated freezing and thawing, and keep in separate containers. Centrifuge instantly before use and prepare 20~100 μM storage solution with RNase-free Water or ddH2O, the average molecular weight of siRNA is 13300.

(2) RNA handling rules should be strictly followed and RNase-free lab supplies should be used for experimental operations to avoid degradation of RNA. Please keep on ice when using this product.

(3) For RNA with fluorescent marker, we use brown centrifuge tube to pack, because the fluorescent marker is sensitive to light, it must be stored away from light.

Before opening, centrifuge the tube. Add sterile RNase-free H2O or ddH2O to prepare a 20 μM stock solution. Aliquot as needed and store at -20°C to -80°C.

Positive controls are verified siRNAs targeting housekeeping or reporter genes, used to assess the feasibility of the experimental methods. Tsingke provides effective siRNA positive controls targeting GAPDH, ACTB, GFP/EGFP.

Negative controls are non-specific siRNAs used to demonstrate the specificity of siRNA effects and can be selected from universal or scrambled sequences based on requirements.

Normally, the results of negative controls should be similar across different groups. Significant deviations may indicate issues with the accuracy of experimental results.

For certain genes, particularly those related to cellular stress or immune response, varying responses to external stress might cause inconsistent gene expression across control groups.

The two most common reasons for unsatisfactory silencing effects are low transfection efficiency and suboptimal siRNA sequence design.

(1) Check Transfection Efficiency: If you are using siRNA for the first time or working with a new cell line, we recommend testing and optimizing the transfection efficiency. If you have already optimized the transfection conditions and the problem persists, consider switching to a different transfection reagent or method, as this might improve transfection efficiency.

(2) Evaluate siRNA Sequence Design: If the transfection efficiency has been improved but the silencing effect is still not satisfactory, it may be due to ineffective siRNA sequence design.

Usually, FAM shows green fluorescence (excitation wavelength 495 nm) and CY3 shows red fluorescence (excitation wavelength 550 nm) after transfection. If no fluorescence is observed, consider the following:

(1) Is the fluorescence excitation wavelength correct?

- Ensure that the excitation wavelengths are set to 495 nm for FAM (green) and 550 nm for CY3 (red).

(2) Were the cells washed immediately after transfection?

- Wash the cells immediately after transfection to observe fluorescence. Delaying this step can result in reduced fluorescence.

(3) Was the siRNA product stored properly?

- Improper storage can lead to siRNA degradation and FAM fluorescence quenching. FAM is particularly sensitive to light, so store it away from light.

(4) Was the transfection performed away from light?

- Operate away from light during transfection. Avoid exposing FAM to white light for extended periods.

(5) Was fresh medium replaced in time after transfection?

- Prolonged transfection times without timely replacement of fresh medium can affect fluorescence. Ensure fresh medium is added promptly post-transfection.

Poor cell health before transfection can lead to increased cell death. Ensure cells are in optimal growth condition, as healthy cells better tolerate transfection reagents. High concentrations of siRNA or transfection reagents can be cytotoxic, so adjusting these concentrations may be necessary. Some transfection reagents are more toxic; consider switching to a less toxic alternative. Low-purity siRNA may contain impurities that affect cell viability; therefore, using high-purity siRNA is recommended. Additionally, prolonged transfection times can exacerbate cell death; ensure timely replacement of fresh medium after transfection.

Low efficiency of cell transfection can occur due to several reasons:

(1) Insufficient siRNA purity may lead to RNA enzyme contamination. Use DEPC-treated consumables and reagents to prevent this issue.

(2) Adjusting the concentration of the siRNA-transfection reagent complex can enhance transfection efficiency.

(3) Serum in the transfection mixture can interfere with efficiency. Prepare the mixture without serum to minimize this effect.

(4) Avoiding antibiotics in the culture medium is recommended as they can inhibit transfection efficiency.

siRNA-mediated RNA interference (RNAi) is a transient phenomenon and cannot be stably propagated through cell generations. Its effects typically do not last for an extended period, and it is generally recommended to complete detection within 3-4 days post-transfection. The optimal detection time varies depending on the cell type and target gene, but it is usually between 24-48 hours post-transfection. It is generally suggested to assess mRNA levels at 24-48 hours and protein levels at 48-72 hours post-transfection.

Transfection efficiency depends on the characteristics of the cells and the transfection method used, and it is not directly related to the siRNA sequence. Therefore, siRNA transfection efficiency may vary across different cell types.

Usually, the package products are guaranteed to interfere with about 70% of the target gene, if the interference effect is about 60%, we also consider the interference to be meaningful. If there is a significant change in the phenotype and function of the cells, it is also recognized in the literature. Generally, the interference efficiency can be improved by increasing the transfection efficiency and optimizing the experimental system.

siRNA acts directly on mRNA, so mRNA level detection is the most direct detection indicator. Many clients believe that the direct result of mRNA degradation should correspond to a decrease in protein content, and therefore protein level assay results can also be used as an indicator of effectiveness. In fact, in many cases there is often a mRNA decline level that does not correspond to the protein decline level. The possible reasons for this are:

(1) Related to the time chosen for the assay. It may be that the decline in mRNA has not yet been reflected in changes in protein amounts or has not reached a level sufficient for detection, so it is generally recommended that protein and functional assays be pushed back slightly.

(2) The translation process of mRNA is very complex, and the expression of genes in the cell must always maintain a balance, and after a certain level of expression of some proteins is sufficient to maintain their cellular function, the expression may be temporarily "turned off" and the transcribed Some mRNAs may not be involved in the process of protein translation, therefore, the down-regulation of mRNA level is not exactly positivelycorrelated with the down-regulation of protein level.

Different cells have different transfection efficiency and different gene expression levels, which are all related to the action efficiency of siRNA, so there is no guarantee that the interference effect is the same in different cells.