I am encapsulating human mesenchymal stem cells in 10wt% polyethylene glycol diacrylate hydrogels. I have to extract total RNA from these cells. So the first step is homogenizing these gels to get the cells out so that the TRIzol can act on the cells. I have tried the following methods of homogenization:
1. Using RNase free Mortar Pestle, I used gels which have been frozen in liquid nitrogen and grind them into a powder using liquid nitrogen. Then I will cover the powder in TRIzol and then once it melts i continued with the normal extraction process.
2. Using RNase free electric tissue homogeniser for ~6minutes.
These methods have not been successful. There is no RNA in the final bioanalyzer electropherographs.
If you can get away from using PEGDA gels, I'd recommend using an HA-based hydrogel I've worked with for a while (HyStem from Glycosan). You can get a fair amount of RNA using a QIAshredder and normal Qiagen RNAeasy kit.
The QIAshredder should work for a pulverized PEGDA gel too. They are pretty cheap, so it may make sense to try them.
I don´t know why you use PEGDA gels and whether you freeze the gels only to crush them to get the RNA out or whether you have other reasons. If you could use Dextran-PEG-based gels, you could dissolve the gel easily with dextranase without having to freeze the gel. You can isolate your live cells by centrifugation and perhaps one wash step to get rid of the rest of the dextranase. You can treat them then as usual for RNA extraction. The advantage of the dextranase is that it does not harm the cells because it is not a protease. The only drawback is that the dextranase digest takes 30 min. If you think your RNA profile will change when cells are isolated this way, you may want to check this with a control culture and RNA isolation. The Dextran gels are from Cellendes (www.cellendes.com) and you can get them in the States from B-Bridge International (www.b-bridge.com). They are called 3-D Life Hydrogels.
I have used PEDGA gels at both 10wt% and 30wt% (MW 3000 and 4600) and have had success getting the RNA of hMSCs out. I did spend a considerable about of time with the automated pestle (after freezing) for each sample (1-2 minutes for 10wt%), however. At first I used the TRIzol method which was okay, but in the end I switched to the QIAGEN RNeasy extraction columns (http://www.qiagen.com/Products/Catalog/Sample-Technologies/RNA-Sample-Technologies/Total-RNA/#&&p=1&s=Relevance). They have both normal kits as well as "micro" kits if the amount of RNA that you're trying to extract is very small. The nice thing about the columns is that they separate the RNA from the annoying PEGDA gel bits in the first step. Although much of the RNA will always remain trapped in the gels (the amount depends on the molecular weight of the PEG that you're using), this seems to be the "cleanest" and most efficient way to get the most out that you can.
One thing to note is that your encapsulation density is quite low, and I think you will struggle to get very much RNA out of your samples. One idea that you could try is to encapsulate at a higher density and then go through the extraction process to see if it's the process that isn't working for you or if it's just your low encapsulation density that is causing the problems. Either way, I think the micro spin columns might help.
Good luck!
Hey thanks for the replies :) @Neven Steinmetz... I am planning on increasing my cells density and performing the extraction using TRIzol followed by spin column purification. Afetr I encapsulate my gels I will free them in liquid nitrogen and then use a mortar pestle to grind them under liquid nitrogen and then add TRIzol. To remove the gel pieces I will centrifuge them and then follow the purification steps. If this does not work then I will try using the QIAGEN kit. Could you tell me the cell density you have worked with? Thanks again for your input.
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