siRNA Transfection Protocol
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How to perform siRNA transfection with Lipofectamine® RNAiMAX protocol. Superior siRNA/miRNA delivery and gene knockdown.
In this video, we will perform an siRNA transfection experiment using Lipofectamine® RNAiMAX reagent.
As always, use good cell culture practices and wear your personal protective equipment. Clean the cell culture hood and work surface by spraying and wiping them down with 70% ethanol.
The day prior to your transfection, seed your cells so that they will be 60% to 80% confluent at the time of your experiment.
The day prior to your transfection, seed your cells so that they will be 60% to 80% confluent at the time of your experiment.
For this transfection experiment you will need:
- Lipofectamine® RNAiMAX reagent
- Opti-MEM® Reduced-serum Medium
- siRNAs diluted to a working concentration of 10 micromolar. We will be using two Ambion® Silencer® Select siRNAs, a BLOCK-iT™ Alexa Fluor® red fluorescent siRNA, and a negative control siRNA
- Five, 1.5 milliliter microcentrifuge tubes in a rack
- A P200 and P10 pipette and appropriate tips
- A marker and a timer
- And a 24-well plate with 60% to 80% confluent cells
We will be following the 24-well plate format of the Lipofectamine® RNAiMAX protocol.
Because we have 4 siRNAs, we will prepare a master mix of RNAiMAX. Add 200 microliters of Opti-MEM® medium and 12 micoliters of RNAiMAX in a tube labelled "Master Mix"
Mix well by vortexing or flicking the tube.
Add 50 microliters of Opti-MEM® Medium into each of 4 tubes and label them 1, 2, Positive, and Negative.
Add 3 microliters of each 10 micromolar siRNA stock to its corresponding tube. Mix well.
Add 50 microliters of the RNAiMAX master mix to each of the siRNA dilutions in tubes 1, 2, Positive, and Negative.
Incubate the complexes for 5 minutes at room temperature.
After the 5-minute incubation, remove your 24-well plate containing your cells from the incubator and bring it to the workspace in the hood.
Add 50 microliters of the siRNA-reagent complex from tubes 1, 2, Positive, and Negative to wells 1 to 4 of the 24-well plate, respectively.
You should have enough volume to run duplicates if desired.
Place your 24-well plate back into the incubator and grow cells for 1 to 3 days at 37 Celsius.
After incubating the cells 24 hours at 37 degrees Celsius, assess the transfection efficiency of the BLOCK-iT™ Alexa Fluor® red fluorescent siRNA using the FLoid cell imaging station or microscope.
To assess gene knockdown use a quantitative method such as Ambion® Cells-to-Ct™ Kit and Real-Time PCR.
For transfection protocols, FAQs, troubleshooting, and tips & tricks visit www.lifetechnol...
Пікірлер: 22
As far as I am concerned, Magic™ mRNA transfection reagent is particularly suitable for mRNA delivery, and has shown outstanding transfection efficiency for both adherent cells and suspension cells.
Can you list the materials (to knockdown a gene) for this experiment so that I can order them, Sir? Thank you so much!
I wish I was watching this video by mistake.
@oprain89
7 жыл бұрын
LOL same here ..but after you in 3 years
@ERICCCAA
5 жыл бұрын
@@oprain89 Same here too! after in 5 and 2 years!
@nicnacioguitar
4 жыл бұрын
6 years and counting
@chandrayeedey7462
3 жыл бұрын
Me after 7 years. 😣
@danieshagovender5817
3 жыл бұрын
Me today 7 years later😥
were cells grown in serum free media for transfection ?
I am wondering the amount of siRNA is correct? for 24 well ,it should be 1ul of 10uM.
@thermofisher
Жыл бұрын
Hello, Thank you for your question. Please contact our technical support team at thermofisher.com/askaquestion for additional information. Thank you!
@Mkvine
Ай бұрын
That’s what their protocol sheet showed. They used 3ul instead of 1 ul in the video. Not sure why.
what is the mechanism of lipofectamin RNaiMax, how is it works with siRNA
@thermofisher
4 жыл бұрын
Great question! The negatively charged nucleic acids associate with the positively charged lipids. The lipids then help shuttle the siRNA across the lipid bilayer. Unfortunately, it is unknown if they just associate of if they form micelles, but the association allows the siRNA to move across the membrane. RNAimax is specialized to work with siRNA and allows for higher potency. It requires less siRNA compared to lipofectamine 3000. For additional technical support, please contact us at thermofisher.com/askaquestion. Thank you!
@xuantrong4467
4 жыл бұрын
@@thermofisher Thank you
I thought this was an ad for a new BioShock game. Disappointed.
were cells grown in serum free media for transfection ?
@skyscraper5910
5 жыл бұрын
idts