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RNA degradation from cells embedded in gels

RNA qPCR collagenase Trizol CTAB

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#1 Lord Apoptosis

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Posted 10 September 2018 - 12:51 AM

Hello, I want to isolate RNA for qPCR from  mouse cells (MC3T3) embedded in gelatin gel, but I am facing issues with RNA degradation
 
First, I was digesting gels with collagenase, and then I have tried both Trizol and CTAB method, based in a reference I attach.
The purity ratios are fine (and better with CTAB, 1.9 for 260/280), but when I check RNA integrity, the RNA is degraded. To digest the gels completely, the time must be over 6 h, and I've done sometimes up to 16 h. I saw elsewhere  that that time can be too long and RNAses can be acting . My first doubt is the source of RNAses, because as far as I know, collagenase doesn't disrupt the cells. Can it be endogenous? And in that case, can they act even from fresh samples, as it happens? My other possiblity now is that the collagenase itself (a powder) is contaminated with RNAses, because I am took care to prepare all solutions with RNAse-free water and DEPC treated instruments.
 
 
I think that I can try shorter times of digestion and then disrupt the remaining gel mechanically. However, in the reference, it is said that digestion was done over 24 hours, although with a different enzyme, and it doesn't seem be affecting RNA integrity. Can someone give me any suggestions, please?
 

 

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#2 OldCloner

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Posted 10 September 2018 - 06:44 AM

I would also be concerned with the collagenase- is the container it came in marked "Molecular Biology Grade" or "DNase-RNase free" or some other phrase to assure you it is appropriate for RNA work?  What is it's purity?

 

When copying a method in a paper it is usually best to use the same supplies they did, in this case Proteinase K.



#3 Lord Apoptosis

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Posted 10 September 2018 - 08:00 AM

Thanks, actually, it's not marked, and that is what makes me consider a source of RNAses. However, I wasn't expecting that to be a problem, as collagenase shouldn't disrupt the cell membrane as far as I know. The cell lysis happens in the CTAB protocol, which contains b-mercaptoethanol, which is known inhibitor of RNAses. But maybe the time with the collagenase is too long and the cells liberate their content when dying, and in that case, if there RNAses it is a problem.

 

It seems the Proteinase K employed in the paper is truly RNase free.


Edited by Lord Apoptosis, 10 September 2018 - 08:03 AM.


#4 OldCloner

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Posted 10 September 2018 - 11:16 AM

I did some searching on Collagenase and found this in the Sigma website- naturally it is trying to sell you a "better" product. But also I noted that there are many types of collagenase and as this quote says, collagenase preps are not pure. Also it says RNAses are released anyway, so they do not test for RNAse contamination.  The quote:

 

Collagenase and Liberase Blendzymes: The differences

Traditional collagenase preparations are isolated from bacterial (Clostridium histolyticum) culture supernatants. These preparations are heterogeneous, containing as many as 30 different enzymes, cellular debris, pigments and endotoxin. Endotoxin levels and variability are the most significant liabilities of traditional collagenase. Liberase enzymes offer many advantages that distinguish them significantly from traditional collagenases. Because each Liberase purified enzyme blend has been specifically formulated per application using response surface modeling, optimal results can be expected in terms of:

  • Increased cell yields
  • Improved cell viability
  • Greater cell functionality

Each Liberase lot has the same enzyme activity specifications. Each lot is tested for endotoxin to ensure consistently low levels. There is no longer a need to perform lot selection or whole batch reservation. Each lot is guaranteed to perform the same in any given tissue dissociation protocol.

Please refer to the Special Interest Site Tissue Dissociation for more detailed information.

RNAse contamination

RNase-free is not a quality control criterion of the Liberase production procedure. During tissue dissociation, cells are disrupted and RNases are released. For this reason, RNase-free quality does not provide additional benefit.

Liberase Research Grade components are highly purified preparations. HPLC chromatograms show there are no other proteins other than Collagenase I and II and the neutral Protease.



#5 OldCloner

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Posted 10 September 2018 - 11:25 AM

Old Cloner again: here is more wit and wisdom from catalog websites, this time Thermo-Fisher. It seems Prot K digests nucleases! maybe that's why they used it.

 

Description

Proteinase K from the fungus Engyodontium album is a nonspecific serine protease that is useful for general digestion of proteins. Proteinase K remains active:

• Over a wide pH range (optimal activity between 6.5 and 9.5)
• Under denaturing conditions (e.g., in the presence of SDS or urea)
• In the presence of metal chelating agents (e.g., EDTA)
• At comparatively high temperatures (optimum digestion temperature is 65°C)

Applications
Removal of endogenous nucleases during the preparation of DNA and RNA; preparation of tissue sections for in situ hybridization.

Performance and quality testing
Endodeoxyribonuclease and exodeoxyribonuclease assays; liquid form tested for absence of RNase activity.







Also tagged with one or more of these keywords: RNA, qPCR, collagenase, Trizol, CTAB

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