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Preservation of human tissue for bacterial mRNA isolation from it later

mRNA Cryopreservation DNA isolation

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#1 Md Jibran Alam

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Posted 16 November 2014 - 05:47 PM

Hi.

I am doing my masters thesis. My research requires isolation of bacteria from colonic mucosal specimen (from colonoscopy) and collecting mRNA from them. But due to delivery problem, I dont have my mRNA isolation kit and cDNA synthesis kit yet. I have collected the specimens in PBS and now kept in 4'C. I cant preserve the tissues now in ethanol due to the reason that the bacteria with the sample will die. Will the bacteria survive and have a functional mRNA if the tissues stored at -80'C with 80% glycerol?

A crucial part of my study is to collect the mRNA from bacteria without culturing. I also want to collect the DNA from the tissues.

 

Please help.



#2 bob1

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Posted 17 November 2014 - 12:20 AM

Keeping things at 4 C is not ideal -this will very likely change the populations of bacteria present in the sample, most of the bugs will be best adapted to living at body temperatures, so cooling like this might well kill of many of them.

 

Gut samples also tend to have a very high level of RNases present, so they often make RNA isolation difficult...You will most likely find that to collect RNA or DNA from the tissues, you will need to store them at -80. There are also solutions out there, such as RNAlater that can be used to store samples at room temp for RNA isolation.


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#3 merlav

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Posted 18 November 2014 - 04:35 AM

Storage in RNAlater and -80C (in rnalater you can store at RT for 24h, 4c 1 week, -20c 1 month and in -80C for long storage) will be a better approach or glycerol and snap frozen in liquid nitrogen.  There is a probability that the samples that you have will not function. My advice stop collecting samples until you have a way to preserve the samples for long time storage. There are some kits that are isolation of bacterial nucleic acids.  


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