How to determine siRNA loading efficiency by Gel electrophoresis?

Samira Naderinezhad @Samira_Naderinezhad

05 May 2018 4 8K Report

Hello everyone!

My colleagues and I performed 2 experiments on gene incubation with nanoparticles followed by electrophoresis and the following results were obtained after imaging:

1-Short time (few seconds) incubations of the siRNA with nanoparticles: All samples moved through the gel and similar bonds of siRNA were indicated for all dilution of nanoparticle and siRNA as well as free siRNA.

2-Long-term incubations (30 mint) of the siRNA with nanoparticles: No bonds were detected even for free siRNA! (It seems there is not any siRNA in the gel)

*The concentration range of nanoparticle/ gene (microgram.micrograme-1): 100:1 75:1 25:1 12.5:1 *Ethidium Bromide-containing agarose gel (2%) were prepared

*Voltage: 50 V *Time: 20 min * Each well contain 1 microliter sample + 3 microliter loading buffer *Sample preparation: 1 microlitter siRNA diluted in 9 microlitter water. Then for each wells, 1 microlitter siRNA sample was mixed with nanoparticles suspension diluted in water.

Thank You

Sivadas VP

Samira,

It seems that after 30minutes of incubation, the siRNA got degraded. You can check that by running 2-3 microlitres of your siRNA/nanoparticle on a 4% agarose gel (NuSieve agarose, preferably). Use RNase-free water and utensils for the experiment. Then, for the bond formation between nano particle and siRNA, your nano particles should preferably be positively charged. Check the zeta potential of your nano particle.

Best Regards,

Siva

Francesco Mainini

Hello,

I would use a 1% agarose gel instead ...furthermore we use heparin to displace the siRNA from the NCs because if the sirna is attached you won't be able to see it (it will be stuked in the well with your NCs).

Usuallly the attachment is very fast with cationic NCs.

I suggest you to alwais use ultra pure distilled water for everything. attach the sirna and take out 2 samples of 10ul each (of sirna + NCs). Treat one sample with 10 ul of heparin (50 mg/ml) for 1 hour at 37 degrees. After the treatment add to both samples the loading buffer (blue) and sybr gold (or what you use to detect the sirna in the gel). You should be able to visualize the siRNA in the well like this.

I hope this helps

Regards

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