Kristi Neufeld Molecular Biosciences Department, University of Kansas, USA

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Maged Zeineldin Molecular Biosciences Department, University of Kansas, USA
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Kristi Neufeld

Molecular Biosciences Department, University of Kansas, USA

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Molecular biology

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1 Protocol published

Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis

Authors: Maged Zeineldin and Kristi Neufeld, date: 11/20/2012, view: 20057, Q&A: 1

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 ...

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Ipsita Chatterjee

bar ilan university

Dear Sir,
1. I found a contradiction in the paper that it is mentioned to take 680 mM of Na2hpo4 9.65g for 1 L solution. I will be highly obiliged if you could please make me understand if the final conc. (680mM) is correct or the weigh? (because it should be 96.5 g for 1L if the final con is 680 mM).
2. I tried this protocol once with final concentration of 680mM, but the solution B is crystallizing on ice, since you have mentioned to do it on ice, i would like to ask you, if you also observe the same problem.
3. And it would be really helpful for me to know approximately what is the concentration of RNA you get by this protocol.

11/20/2018 1:36:07 AM Reply

Maged Zeineldin

Molecular Biosciences Department, University of Kansas, USA

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!

2018-12-17 05:37:07

Ipsita Chatterjee

bar ilan university

and if you could let me know the RIN of this protocol

2018-12-17 02:54:51

Ipsita Chatterjee

bar ilan university

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?

2018-12-17 02:54:04

Maged Zeineldin

Molecular Biosciences Department, University of Kansas, USA

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/15596026
or TriZol followed by DirectZol for RNA extraction
https://www.zymoresearch.com/direct-zol-isolation-kits

2018-11-23 06:35:36

Ipsita Chatterjee

bar ilan university

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

2018-11-23 06:26:09

Maged Zeineldin

Molecular Biosciences Department, University of Kansas, USA

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.

2018-11-23 06:18:43