My work is testing the nanofiber material. I have to seed the cell on nanofiber and then check the gene expression, so I have to isolate the RNA from the cell on nanofiber. Usually, I use the cell scraper to detach the cell and then use trizol. But here is nanofiber, is it ok to use trizol instead? or any recommendation else about others method? Thank you.
RNA can be chemically extracted by trizol method cell is bound by memberane and this could be easily ruptured by freeze thaw method
I've isolated RNA from eukaryotic cells on nanofibers. The process was to add RNA later from Ambion, let it sit overnight at 4C, then spin down cells with nanofibers. Remove the RNAlater from the pellet, and add buffer RLT from a Qiagen RNAeasy kit. Cells will lyse. Make sure you add the correct amount of good 2-mercaptoethanol immediately before using the RLT buffer. Transfer the lysed cells with nanofibers in RLT to a bead beater instrument and homogenize the DNA to approximately 500-800 bp by beadmilling. Keep your sample chilled during this process. Then add 1 volume of RNase-free 70% EtOH slowly with mixing. Your sample can now be loaded directly onto a Qiagen RNAeasy spin column and the RNA purified by following the RNAeasy kit protocol. The nanofibers will remain on the column.
Hi
https://www.ncbi.nlm.nih.gov › NCBI ›
https://genomebiology.biomedcentral.com/articles
1. To passage cells from nanofiber scaffolds to other culture ware the cells can be treated with trypsin, accutase, or other appropriate enzyme as if they were on conventional culture ware. Avoid using cell scrapers. Instead, gently dislodge the cells by careful pipetting and tapping on the culture plates
2. Or you can also incubate the nanofibers scaffold with trypsin at 4C overnight so cell got detached.
3. Or Incubate with Trypsin for 30 min and then collagenase I for 1 to 3 hours or try try a mix of collagenase and dispase form 30 min to 3 hours.
4. Or you can use gentle sonication for 5-15 minutes to detach cells from nanofibers
5. To prepare cell lysates the cells can be treated with the appropriate lysis buffer directly on the wells. Aqueous buffers without chaotropic agents (urea, guanidine HCl) will not affect the fibers.
6. Lyse the cells for 20 min on ice and scrape the nanofibers with a pipette tip to prepare a total lysate in the well. Collect the lysate with a pipette and transfer to a suitable tube
7. Centrifuge the tube for 10 min at 10,000g to remove insoluble fibers and cell debris
8. To extract RNA/DNA, cells can be collected in tubes as indicated above or the RNA extraction solution (eg, Trizol or similar) can be added directly to the wells and recovered by pipetting (proceed as if using conventional cultureware). This will melt and dissolve the fibers, therefore, the cell/fiber suspension must be centrifuged (10 min at 10,000g) to remove insoluble debris before continuing with RNA/DNA extraction
Thank you all the answers. Your answers inspired me about works.
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