Although the Ribogreen Assay works very very well, you could always just use a nano drop to quantify RNA concentration. You could do this by lysing open your nanoparticles, spinning down the nanoparticle fragments via ultra-centrifugation, and reading the siRNA concentration in the supernatant.

Stephen: Is there any need to disintegrate the encapsulated siRNA? I tried both and there was no big difference by nanodrop.

In my opinion, Ribogreen is more sensitive than nanodrop. Any thoughts?

Venkatesh,

You are 100% correct, Ribogreen is more sensitive than nano drop. In addition, ribogreen does not get effected by other components in the solution such as lipid (which is a pain when reading absorbance). To answer your question, no there is no need to disentegrate the encapsulated siRNA before reading. However, you must make sure you remove all lipid before performing the absorbance reading with the nanodrop because this will cause interference. What I would do first is remove all un-encaspulated siRNA (via dialysis or ultracentrifugation). You could quantify this with the nanodrop to determine the un-encapsulated concentration. Then use Triton to permeabilize/disrupt the liposomes and free the encapsulated siRNA. Following this you could use ultracentrifugation to pellet the  lipid, and read the supernatant for encapsulation efficiency. From this point, the conservation of mass can be used to determine the exact amount of encapsulated siRNA.

As you can see this is a lot more work, but it should yield the same encapsulation efficiency as the Ribogreen assay.

Thank you Stephen for your inputs. I like the last two lines. That is why, I am with Ribigreen :-)

Stephen

how can i disrupt the liposomes with Triton? how much is the concentration should be and how much should i use and for how long?

also if i want to use dialysis to remove the un-encapsulated siRNA, does the buffer that i am dializing with need to be RNAse free as well?

thanks

Mohammad,

I typically use 0.05 - 0.1% Triton (vol %) in PBS to disrupt my liposomes in combination with agitation via a vortex mixer for 5-10 minutes (a bath sonicator can also be used to completely lyse the liposomes). I will upload my full text for my JBN paper in the upcoming two weeks or so when I get the final form; I have the protocol explained in detail in that paper. For dialysis, yes you need to make sure the dialysis buffer is also RNAse free.

Good Luck :)

Stephen

thanks a lot.

i need to disrupt the particles in the 96 well plate for the Ribogreen assay so i cant use vortex or bath sonicator so is shaking would be enough?

also how much volume of Triton x should i use in each well bearing in mind that each well contain 100 micro-liter of the siRNA solution, 3 micro-liter of my particles and 100 micro-liter of the ribogreen solution?

thanks

Mohammad,

Since you cant use a vortex or bath sonicator, I would increase the concentration of triton to 0.5% (vol %; or approx. 11 micL stock triton per well of each 96 well plate of about  214 micL total volume). However, I would make a solution of triton in PBS and add this to each well (it takes a while for triton to dissolve). In addition, please note that triton does interfere with the ribogreen assay (it reduces the output fluorescence). Therefore I would perform this entire experiment with a low (0.05 vol%), medium (0.1%), and high concentration (0.5%) of triton and compare.

some folks use RNase treatment to chop the siRNA that was not encapsulated, and measure the difference. i prefer the functional readout- test efficiency of these complexes for delivery and mRNA knockdown, dont worry much about the % of siRNA encapsulated inside the particles. its especially taking into account that some siRNAs are attached to the surface of the particles

Hi there,

I will measure the encapsulation efficiency of SiRNA in DOTAP and DOPE liposome. I want to do it using Nano-drop and not the Ribogreen assay. The comments mentioned above is good. But I want to have the whole protocol(including the calculation and step wise procedure) for the SiRNA encapsulation measurement most importantly, the time and speed needed to ultracentrifuge the nanoparticles. I am kind of new in this area. So, any help will be appreciable.

Any good reference which used nanodrop to measure the SiRNA will be appreciable.

Thank you.

Ribogreen is the best method. Some times it may be harder to release the siRNA from the liposomes completely, in particular while using multivalent cationic lipids. If Triton X100 is not helpful, in releasing the siRNA completely then chloroform extraction method can be followed as described by someone above. Otherwise, one can use curcumin which can dissociate the the siRNA from complexed lipoplex. Ideally 60 ug/mL of curcumin is sufficient to dissociate the siRNA, although it may need some optimization. More details can be found in the below link::

https://www.hindawi.com/journals/jnt/2016/7051523/

Hello,

Are these methods same for nanoparticle too?

please suggest a suitable method for evaluation of siRNA from the polymeric nanoparticle.

Thank you