I am going to isolate RNA from Buccal swab. I have experience in extracting RNA from frozen blood and PBMC. But, I wanted to know which RNA extraction kit will be appropriate? Can I extract sufficient amount of RNA? What kind of challenges I need to face?
With that small of tissue, I would recommend Trizol reagent (http://products.invitrogen.com/ivgn/product/15596026) . If you use a kit, you will use a lot in the process and you will not have a lot of starting material with a buccal swab.
I use trizol to extract RNA from microscopic sections captured by laser dissection, and achieve high quality RNA used for real time RT-PCR. This should work well for your process as well.
Good luck,
~Adam
With that small of tissue, I would recommend Trizol reagent (http://products.invitrogen.com/ivgn/product/15596026) . If you use a kit, you will use a lot in the process and you will not have a lot of starting material with a buccal swab.
I use trizol to extract RNA from microscopic sections captured by laser dissection, and achieve high quality RNA used for real time RT-PCR. This should work well for your process as well.
Good luck,
~Adam
I agree completely with Adam. Trizol (Invitrogen) works best for small starting material
As the others indicated, from a standard swab your yield will be very low. This company has a new system for collection and stabilization of high yield buccal cell RNA that looks like it can be combined with TRIzol, or some less dangerous kits like MasterPure: http://www.mawidna.com/products/iswab-rna-collection-kit/
I was looking for buccal swabs for RNA isolation on the net.
Which do you recommend? Can one collect samples using cotton swabs (eg http://www.ebay.com/itm/252582255333), put them in RNA later and isolate RNA?
Has anyone other idea how to obtain samples?
Dear Josipa Vlainic!
RNA extraction from the cotton buccal swab is my usual work in my research.
By using 300 µl of D-MEM (Low glucose) for each sample, I make homogenate then using 100 µl of this homogenate solution and using Trizol method, I get good quality RNA.
Dear Wazir Ahmad Habibi!
I am hoping to collect RNA from vaginal swabs, and I had the same kind of question. Do you think it is sufficient to use just sterile cotton swabs (eg http://www.ebay.com/itm/252582255333) or should I use swabs made specifically for cell collection (eg https://www.amazon.com/Whatman-WB100035-Omni-Buccal-Sterile/dp/B00394FIQA)? Also, if I am collecting samples in a location where I cannot immediately extract them, is it ok to store the swabs at -80 as is? Or should they be placed in some kind of stabilizer/RNAlater and then frozen?
Thank you so much for any advice!
Dear Rachel,
I would not recommend freezing swabs/cytobrushes without a stabilizing agent. lining cells/epithelial cells tend to break open after thawing and thus releasing cellular leading to RNA degradation, almost immediately. Using RNAlater might help, but the quality and yield of the RNA will be affected substantially. Having said that, we have developed a simple collection technology that can stabilize cells intact at room temperature for up to 4 weeks. We have tested buccal cells which are quite similar to vaginal epithelial cells. Actually, the collection system is designed to recover all cells from several swabs-cytobrushes and then stabilizing them in a concentrated format, with out the need to send back the swabs witht he sample. You can either keep at room temperature for few weeks or freeze at -20C or -80C until you need to use them, you have up to 3 thaw and freeze cycles/sample. I have attached some data for your review. I hope this helps
Agnieszka Szemiel
to clarify further iSWAB-RNA v2 is a sample collection and concentration system from swabs and not an extraction kit.For microbial collection we have optimized iSWAB-Microbiome to simplify collection from different sources, including nasal swabs. This collection system you can isolate both microbial DNA and RNA from the same sample (https://www.youtube.com/watch?v=2mIdfHwoeMY&t=3s). Unfortunately, we have not tested RNA recovery from swabs after being stored in RNAlater. Having said that, I think your best option for isolating viral RNA from RNAlater stored samples using mag bead based kits such as MagMAX-96 AI/ND Viral RNA Isolation Kit (Cat #AM1835) or kits that utilize Ply d(T) tail to enrich for viral RNA. The paper link has more details on this and I hope this help address your question better:
https://www.vanderbilt.edu/vinse/am506374t.pdf
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