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Homogenation of Mouse Spinal Cord Tissue for RNA Extraction


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#1 BSM

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Posted 01 September 2009 - 02:35 PM

Hey everyone,

I am looking for an effective way to homogenize 20-30mg sections of spinal cord tissue in preparation for a trizol extraction. I have already performed this procedure weeks ago and ran into some unforeseen issues while doing it. I initially was planning on using an electric homogenizer in a 15ml conical tube but I realized that the rotor was too big and there would be too much loss of tissue when only using 200-300 microliters of Trizol for each sample. I then decided to try a 1ml dounce homogenizer but, yet again, there was going to be too much loss of tissue trying to remove the homogenate from the dounce. There was also some ugly foaming. I last tried using a handheld device (like a handheld homogenizer) that has connecting plastic pestle tips that fit into 1.6 or .6ml eppendorf tubes and uses the vibrating motion to homogenize the tissue. This method also did not work because the tissue did not seem to break up enough inside the tube because the vibrations along were not strong enough. I wound up with a fair amount of RNA after the extractions but I did notice some degradation after running the RNA on a gel. This was likely due to the tissue samples sitting in trizol for too long when I was trying to figure out a better way to get homogenation.

Is my only other option to find a handheld electric homogenizer for this small of sample?

Suggestions?

Thanks!

Edited by BSM, 01 September 2009 - 02:36 PM.


#2 eberthella

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Posted 01 September 2009 - 05:25 PM

Stick the animal (preferable dead) in a blender (Waring type) and homogenize with enough buffer to cover. QS to 500 or 1000 ml. Worked well for microbial counts.

#3 gfischer

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Posted 02 September 2009 - 08:11 AM

I assume the vibrating handheld device you refer to is a sonicator. I'd be careful with that as they generate quite a bit of heat in small volumes, which might damage the RNA. A glass conical or dounce homogenizer should work fine for your purposes. Also, there's no need to use such a small volume of Trizol, since you can adjust your final RNA concentration by changing the volume of water/TE buffer you resuspend the RNA in after precipitation. I'd recommend using 1mL of Trizol and resuspending the RNA in 50-75uL of DEPC water after precipitation.
Above all things, if kindness is your king,
then heaven will be yours, before you meet your end

#4 phage434

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Posted 02 September 2009 - 08:21 AM

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