We are culturing cells inside a 3D PLGA electrospun scaffold. Currently, we are using Qiazol to isolate the RNA, and we found that the Qiazol dissolves the PLGA scaffold upon contact. And we also found that the RNA subsequently extracted via mini Qiagen kit in this manner was degraded when compared to that obtained from 2D controls. Has anyone successfully extracted RNA from a PLGA scaffold?
We also tried to trypsinize the cells down from the scaffold, however the cell yield is very low leading to very low concentrations of extracted RNA.
in your RNA isolat kit you have lysis buffer, use that to lyse your cells from inside of your scaffold. Be quick, RNA is not stable.
How are you assessing RNA degradation?
And how are you assessing cell yield after trypsinization? It may be that you simply don't have many cells.
If in doubt, my go-to piece of advice (before any more technical troubleshooting) is to use more Trizol/Qiazol. It doesn't really hurt to use MORE than you need (though it is marginally more expensive), but it can be really quite detrimental to use LESS. For example, I typically use double or triple the suggested ratio of qiazol:tissue for fatty tissues like brain. If there is something in the dissolved PLGA that degrades RNA you can minimize this by using more Qiazol: simple dilution principle.
Yes, the trypsinization isn't effective as most of the cells still remain inside the scaffold trapped by the fibers and only those on the surface are removed from the scaffold resulting in a low cell yield(when I see the cell pellet after centrifuging) and in turn a very low concentration of RNA. However, I wonder why the RNA degrades when the PLGA scaffold is dissolved in Qiazol. This should be a better method as all the cells including those inside the scaffold should contribute to the RNA giving much higher concentration.
in your RNA isolat kit you have lysis buffer, use that to lyse your cells from inside of your scaffold. Be quick, RNA is not stable.
- Badges
- Science method