how to optimize sirna transfection time course

Generally, transfection optimization could be achieved by transfecting cells with siRNAs targeting endogenous genes such as Lamin A/C and GAPDH and then analyzing their expression by RT-PCR or Western blotting. Alternatively, assays using reporter genes including GFP and luciferease, which are faster and more quantitative than Western blot, facilitate time-consuming optimization experiment.

Full Answer

How can I optimize siRNA transfection?

Dilute siRNA using the manufacturer’s recommended buffer. Alternatively, use 100 mM NaCl in 50 mM Tris, pH 7.5, made with RNase-free water. Do not use water alone to dilute siRNA, as this may result in denaturation of the siRNA. siRNA Concentration. siRNA used for transfection should be highly pure, sterile, and the correct sequence.

What is the incubation time after adding siRNA to the cell line?

In our experiments, a single transfection of 5 nM Silencer Select siRNAs achieved >80% knockdown that lasted 5–7 days post-transfection, then progressively diminished. Higher siRNA concentrations did not result in stronger or longer-lasting …

Can siRNAs be transiently transfected?

for the delivery method of choice (such as transfection reagent, or electroporation) and taking measures to test and optimize the conditions best suited for the cell line or culture selected. Review protocols using DharmaFECT™ transfection reagents or Accell™ siRNA delivery. Table 1. Recommended siRNA resuspension volumes and concentrations

How much siRNA is needed to transfect HeLa cells?

May 16, 2013 · Of course, after a reagent is chosen it’s best to take time to optimize parameters such as quantities of reagent (use the least that gets the job done) and siRNA, timing of the transfection and how long to wait before testing for effect. This is best done by optimizing around a combination of RT-PCR and a downstream assay, says Yun.

How do you optimize a siRNA transfection?

9 Tips for Optimal siRNA TransfectionUse the most appropriate siRNA concentration. ... Prepare a suitable siRNA stock solution. ... Transfect healthy cells. ... Check serum quality. ... Know the target gene in and out. ... Always use positive and negative controls. ... Follow up the transfection reagent protocol.More items...•Aug 3, 2012

How would you increase your transfection efficiency next time?

Here are some tips that may help you improve your transfection success.Transfect healthy, actively dividing cells at a consistent cell density. ... Transfect using high-quality DNA. ... Optimize the amount of DNA used to transfect cells. ... Optimize the transfection reagent:DNA ratio.More items...•Jul 15, 2016

How can I improve my siRNA knockdown efficiency?

Be Consistent When Conducting Experiments.Select Appropriate Order of Transfection.Use Healthy Cells at the Optimal Density.Choose the appropriate Culture Media and Culturing Conditions.Use High Quality siRNA at the Lowest Effective Concentration.More items...

How much siRNA should I use for transfection?

1-30 nM siRNAIn general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point).

How do I optimize my transfection?

Other factors to consider during optimization are: (1) nucleic acid purity (higher is better and must be endotoxin-free), (2) nucleic acid concentration (needs to be determined precisely), (3) complex formation time (typically 15-30 minutes but can also be dependent on application), (4) cell confluence (avoid too low ...Sep 19, 2018

What causes low transfection efficiency?

Confluency and replication state Cell confluency and replication stage are factors intrinsically linked to each other that affect the efficiency of your transfection protocols. This is because cells that are actively dividing take up DNA more readily than stationary phase cells.Jun 28, 2018

How long does siRNA knockdown last?

5–7 daysThe 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 does siRNA affect gene expression?

Once the single stranded siRNA (part of the RISC complex) binds to its target mRNA, it induces mRNA cleavage. The mRNA is now cut and recognized as abnormal by the cell. This causes degradation of the mRNA and in turn no translation of the mRNA into amino acids and then proteins.

What is the main difference between miRNA and siRNA?

The major difference between siRNAs and miRNAs is that the former inhibit the expression of one specific target mRNA while the latter regulate the expression of multiple mRNAs. A considerable body of literature now classifies miRNAs as RNAi molecules.

What concentration of siRNA should I use?

In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point).

How is siRNA concentration calculated?

What is your target concentration to treat the cells? Like, if you want to treat 100 nM concentration, the calculation will be ((100 nM/20 uM)*500 uL) = ((100 nM/20 x1000 nM)*500 uL) = 2.5 uL (of stock siRNA).Jan 31, 2019

How does siRNA knockdown work?

Through the activity of several proteins (discussed below), targeting of a cellular mRNA by short, anti-sense nucleic acids (siRNAs and shRNAs) results in its subsequent degradation. This, in turn, blocks further expression/accumulation of the proteins, leading to a decrease in its levels, and eventual knockdown.

Why do you transfect a non-targeting or non-sense siRNA control sequence?

Transfect a non-targeting or non-sense siRNA control sequence to verify that the gene expression knockdown or phenotype is attributed to the gene-specific siRNA. Additionally, targeting a gene with multiple siRNA sequences ensures that the resulting phenotype is not due to off-target effects.

What is the optimal siRNA concentration for transfection?

Depending on the type of experiment, the optimal final siRNA concentration for transfection is typically within the range of 10-50 nM. As a starting point, we recommend 25 nM siRNA (final concentration in well).

How long to incubate siRNA?

After mixing the siRNA and transfection reagent, incubate to form complexes for 15-30 minutes at room temperature, before adding the mix to your cells. Transfection efficiency may decrease if the complex formation exceeds an hour.

Why is it important to maintain a similar passage number between experiments?

Maintain a similar passage number between experiments to ensure reproducibility. A low passage number can make cells more sensitive to transfection whereas a high passage number can render cells refractory to transfection.

How much density is needed for transfection?

Cells need to be passaged frequently and the transfection should always be carried out under the same culture conditions. Usually a high cell density of around 70% is needed at the time of transfection. However, this depends on the cell type and should be determined for each experiment. 8.

What is the difference between positive and negative control?

Positive control: A known siRNA to give a high knockdown of the target of interest. Negative control: A non-silencing siRNA helps to identify non-specific changes in gene expression.

Do transfection reagents need serum?

Most transfection reagents require a serum-free medium for the initial dilution of the siRNA complex. If sera are added to the cell culture while the transfection is carried out, the quality/lot might also affect the experiment.

Does siRNA bind to introns?

siRNA should not bind to introns. No sequence that shows homology to other coding sequences. 4. Working with a new target/siRNA/cell line. Be prepared to run multiple test transfections in order to optimize the best conditions.

What is siRNA transfection?

siRNA transfection is a powerful tool to understand underlying mechanisms in gene regulation and molecular pathways. Transfecting siRNA with a high efficiency while minimizing off-targets and side effects will not be a challenge for you anymore thanks to these tips. Use the most appropriate siRNA concentration.

How long after transfection can you measure gene silencing?

Check the half-lives of the protein and mRNA of interest and measure gene silencing accordingly 24 to 96 hours after transfection. Analyze gene silencing at both mRNA and protein levels. Always use positive and negative controls. Use an siRNA against a housekeeping gene (GAPDH, cyclophylin B) as a positive control.

Can antibiotics inhibit reagents?

Some reagents are inhibited by serum or antibiotics, while others may be used in serum- and antibiotic-containing medium, hence reducing the risks of toxicity and the number of steps in the protocol. Check the recommended cell confluency. Perform an appropriate read-out. Test different antibodies if possible.

Does serum reduce silencing?

Some serum lots may drastically inhibit transfection efficiency, hence resulting in lower silencing efficiency. Check transfection efficiency of different serum lots before purchasing a new batch of serum. Know the target gene in and out. Design the siRNA sequence as efficiently as possible by using several algorithms.

What is the duration of silencing after a single siRNA transfection?

In readily transfected cells treated with potent and effective siRNAs such as the new Ambion Silencer Select siRNAs, near-maximal silencing can be achieved for 5–7 days. Figure 1 shows data from a representative experiment. Four different Silencer Select Pre-designed siRNAs were transfected at 5 nM into HeLa cells.

Can silencing be prolonged by raising the siRNA concentration transfected?

No, raising the siRNA concentration from 5 nM to 50 nM did not improve or prolong silencing (Figure 2). Presumably, the RISC is saturated upon efficient transfection of a highly potent siRNA at 5 nM; excess siRNA could potentially be rapidly degraded, sequestered, or excreted from the cell.

Can prolonged silencing be achieved by repeated siRNA transfections?

Repeated transfections can prolong silencing in some cases, however, we have seen variable results with this approach.

Conclusions

In our experiments, a single transfection of 5 nM Silencer Select siRNAs achieved >80% knockdown that lasted 5–7 days post-transfection, then progressively diminished. Higher siRNA concentrations did not result in stronger or longer-lasting knockdown, but are likely to cause off-target effects.

siRNA-specific reagents?

Long before the discovery of siRNA, nucleic acids were being transfected into cells using carriers such as CaPO4, polysaccharides like DEAE-dextran, various lipid formulations, peptides and combinations thereof.

Search, ask and test

So of all the reagents—siRNA-specific or generic, lipid-based or not, a household name or a hot new product, premium or econo-priced—what should you use? It depends in part on the cell type and the species, the downstream assay and whether it’s a co-transfection, how much legwork you’re willing to put in and a few other considerations.

What to look for

There are two main things to watch for when testing a transfection reagent: Does it allow the siRNA to get into the cell? And is it relatively free of negative effects? If the answer to either of these questions is “no,” try another.

Final considerations

Finally, check with the vendor to make sure the reagents you choose are compatible with your assays and workflow. Some protocols require serum-free medium, for example, or changes of media. And although these types of incongruities may not be insurmountable, if two reagents are otherwise equal, you’ll know which one to choose.

How long does it take to harvest DNA from a cell?

Usually 24-72 hours, depends on the cell type, transfection reagent , and your target. It is always recommended to determine the best reagent and DNA concentration, and the best time point for harvesting. Cite.

How long does it take for 293T cells to work?

in case of 293T cells ,for 6 well plate system it you started with 5*10 5 cells , 48 h to 72 hours should work. But it depends on many factors, including transfection reagent, scale and type of cells

Protocol Optimization

See more on ptglab.com

Rnase-Free Environment

Designing siRNA

siRNA Controls

Validation of siRNA Data

Cell Culture

Serum Or Serum-Free Medium?

Antibiotic Or Antibiotic-Free Medium?

Duration of siRNA Silencing

Futher References