GWD

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Basically looking to get a single cell suspension from whole tissues (an array of types) to use for protein and mRNA extraction...

I think in the past when I've done this, I did a collagenase digest for some time, and then extracted for proteins using a cell lysis buffer.

Anybody got any references or protocols that have worked well for them (what tissue type?)
 

Sulfinator

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When I isolated fibroblasts from whole mouse embryos, I first macerated the tissue (whole embryo) with a razor blade, and then used trypsin to breakdown all the extracellular matrix. This gave me a cell-rich soup. After a few passages, the fibroblasts had outgrown everything else and it basically became a fibroblast culture. I wonder if you couldn't do the same thing with your tissues, just without culturing the cell-rich soup.
 
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I did this quite a bit with various mouse tissues to get RNA. My initial method was to harvest the tissue as quickly as possible and throw it right into liquid nitrogen...obviously everything needs to be treated with an RNAse inhibitor. I'd use a mortar and pestle to turn it into a powder and then use my standard lysis buffer for RNA or protein with inhibitors.

The second method I found to be MUCH better was using a commercial tissue homogenizer column. It is a small 2ml tube with ceramic beads inside that goes onto a vortex of sorts. I got fantastic tissue homogenization in about 20 seconds right in my RNA lysis buffer with inhibitors. Of course with all the friction, you need to keep everything on ice and use the homogenizer in a cold room. This gave me great quality RNA for running affymetrix arrays.

The final method I've tried and hated has been the little wand style tissue homogenizers. (they look like little drills and spin around) I never had much luck at all with these. There were always annoying clumps of tissue.

Long digests with things like collagenase, trypsin etc need to be done at temp and will reuslt in crappy degraded RNA or protein. I only use it now for getting cells for plating out.

For protein, the most crude way is to just put your tissue chunk on a cell strainer placed into a 50cc conical tube. Use the inside plunger of a 5cc syringe to mash it through the strainer periodically rinsing with ice cold PBS. After you mash it all/rinse it all through, just spin down the cells and resuspend in your standard protein lysis buffer. I use a 1% Triton buffer with lots of inhibitors.

What ever you do...especially for RNA: Work fast. Keep it cold. Use pleanty of inhibitors. Affy arrays are $$$ and if you start with crappy RNA, you will get crappy results. If you institution has a molecular biology core facility, call them up and see what they like to use. Maybe they will even let you have some homogenizer columns for free to give a try...usually they will have demo kits.
 

GWD

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Thank you EM... that is very helpful.

I have also used the cell strainer method before to some success. I was worried about possibly needing to use collagenase, because many of my tissues I'm expecting to be fairly fibrous (trachea, artery, bladder, ileum, jejunum, duodenum, and rectum for starters), but you bring up a good point about the temp degrading RNA/proteins.

I'm planning on using the protein for Westerns, and the RNA for qPCR, btw.

Can you give me a little more information on those homogenation columns? Sounds like they may have some potential. Vendor? Do you need a special piece of equipment that does the vortexing?
 

Charles_Carmichael

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I did this quite a bit with various mouse tissues to get RNA. My initial method was to harvest the tissue as quickly as possible and throw it right into liquid nitrogen...obviously everything needs to be treated with an RNAse inhibitor. I'd use a mortar and pestle to turn it into a powder and then use my standard lysis buffer for RNA or protein with inhibitors.

The second method I found to be MUCH better was using a commercial tissue homogenizer column. It is a small 2ml tube with ceramic beads inside that goes onto a vortex of sorts. I got fantastic tissue homogenization in about 20 seconds right in my RNA lysis buffer with inhibitors. Of course with all the friction, you need to keep everything on ice and use the homogenizer in a cold room. This gave me great quality RNA for running affymetrix arrays.
My lab used to do the first method for a while but just this past year, we got the tissue homogenizer machine. I cannot remember who the vendor was off the top of my head, but I definitely remember one of those salespeople coming in and showing us how to work the machine. I'm pretty sure it's a Qiagen TissueLyser, but I can't be 100% positive. I'll try to remember to make a note of the vendor when I'm in lab tomorrow. You don't need a separate machine to vortex the tubes. You place the beads into the tubes containing your tissue and then, place the box containing all your tubes (with beads in them) in between two arms on the machine. Then, you can adjust the frequency of the vibrations, etc.

The tissue type we use this for is brain. We do a ton of assessment of hippocampal cytokine mRNA expression, etc.
 

GWD

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You don't need a separate machine to vortex the tubes. You place the beads into the tubes containing your tissue and then, place the box containing all your tubes (with beads in them) in between two arms on the machine. Then, you can adjust the frequency of the vibrations, etc.
So you DO need a special machine to use this system?

I'll eventually being using brain as well, but it seems much more straight forward since it is such a soft tissue. I'm most concerned about the fibrous, muscled tissues I am using.
 
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I've used the LN2 and commercial tube methods on otherwise tough tissues to homogenize like muscle and skin. Before doing arrays, I checked for expression of my transgene in just about every tissue you can think of. The LN2 method works. It just isn't nearly as nice as the commercial device. If you can't persuade your PI to buy the bench top machine the tubes go into and you don't have a core facility with one, I'd try to LN2 method. Give it a shot and see what you get as its super cheap and might be your best option for RNA from very tough tissues like skin and muscle.

LN2 method: Extract your tissue and put it straight into a mortar with just enough LN2 to quickly freeze it and completely evaporate. Make sure you treat your mortar/pestle with an RNAse solution like "RNAse Away." When the tissue is rock hard, turn it into a fine powder...should only take a few seconds. Ta da.....now you have some tissue on which to use your lysis solution. I'd first use ice cold RNAse free PBS to rinse your tissue out of the mortar and into a 1.5ml tube, spin it down cold and then lyse.

Check your RNA using whatever method you prefer and see what you've got. Worst case senario, your out 1ml of LN2 and about 10 minutes of your time.

Mortars are cheap but make sure you get one that can withstand the cold of LN2. Be gentle as most mortars will break if you hit them hard with the pestle after they have had LN2 in them. Also...the frozen tissue pieces tend to "jump around"...be gentle.