Abstract
The gastrointestinal (GI) tract is lined by a single layer of epithelial cells which function in secretion, absorption, and digestion. In addition, most GI tract tumors develop from epithelial cells (carcinomas). This protocol describes isolation of the surface epithelium from the underlying stroma, muscular layer and submucosa in the GI tract. In this protocol, epithelial cell adhesions are weekend by chelating Ca +2 ions followed by mechanical separation of the cells by vortexing. Analysis of protein levels and gene expression patterns in isolated epithelial cells versus whole GI tissue minimizes the potential for confounding contributions from contaminating stromal cells.
Keywords: Intestine tissue, Colon tissue, Cell isolation, Protein, Mouse
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was modified from a crypt isolation procedure reported by Kulkarni and Yielding (1985). This work was supported by RO1 CA10922 from the National Cancer Institute and P20s RR15563 and RR016475.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Thanks for the questions!The weight is the correct value (9.65 g/L) and you should not see any precipitate with this concentration. For the RNA yeild, you should be able to get at least 20-ug RNA by following this protocol.
Hi, Sorry for bothering you one more time, I am isolating the RNA with QIAGEN kit, only from large intestine, Last time i barely got any RNA. Could it be beacuse i used a higher concentration of NA2Hpo4? I m guessing you are also isolating RNA seperately from small and large intestine , and for each you are getting minimun 20 ug yield ? Please if you could suggest anything
I assume this was because using the higher concentration. I believe when using the correct concentration of Na Phosphate you will get good amount of RNA. Small intestinal pieces yield more RNA than the colon but I used to get al least 20-ug from the colon as well. However, I have not used Qiagen for RNA extraction in this protocol. I used to use Trizol https://www.thermofisher.com/order/catalog/product/15596026or TriZol followed by DirectZol for RNA extractionhttps://www.zymoresearch.com/direct-zol-isolation-kits
Hi, sorry to disturb you one more time, at the end of the procedure there is the centrifuge, in the paper you mention both 1000 g and 1000 rpm for 10 minutes, may be if could please tell me its 1000 rcf (g) or 1000 rpm for 10 minutes?
and if you could let me know the RIN of this protocol
Thanks for the questions!You can use either (Xg) or rpm. I have not run the RNA on a bio-analyzer to give you the RIN. However, I ran it on agarose gel and it looked good to me.Good luck!