Effect of triton-X concentration on RNA stability

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Effect of triton-X concentration on RNA stability

Postby SyntonicC » Jun 24 2015 9:34 am

I am attempting to isolate RNA from the post-synaptic density of rat neuronal cells. I am specifically interested in mRNA here that is being translated actively by the ribosome.

The procedure I am using to isolate this cell fraction is approximately as follows:

Homogenize brain tissue >
Filter homogenate through 100 um filter >
Filter homogenate through 5 um filter (now have synaptoneurosomes) >
Incubate in 1% triton-X + 50 mM Tris for 10 min at room temp >
Spin down sample (14k rpm, 10 min, room temp)

The pellet here is the "triton-insoluble" fraction, a crude prep that mostly isolates the post-synaptic density from neurons. The supernatent is the "triton-soluble" fraction which contains axons and dendrites. To lyse the cells I apply RIPA (containing 0.1% SDS and 1% triton-X).

To extract the RNA I use the following kit: https://norgenbiotek.com/product/rnapro ... n-plus-kit

When I check my samples via BioAnalyzer, the peaks I am getting are indicative of rRNAs (very litle), tRNAs, and perhaps some small RNAs (30-180 nt). In other words, no mRNAs were detected.

My questions:
1) Is it possible that triton-X is disturbing RNA-protein interactions and the RNA is ending up in the soluble fraction (I am checking the RNA concentration of the soluble fraction today). I have been searching for data on this today but couldn't turn up anything.

2) Is it possible that the triton-X concentration in RIPA is insufficient to lyse the triton-insoluble pellet? This would mean that the RNA is trapped in the unlysed cells.

Two other possibilities are that there really is no RNA there or perhaps the kit I am using is somehow affected by triton-X.
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Re: Effect of triton-X concentration on RNA stability

Postby relaxin » Jun 24 2015 10:42 am

It seems to me that the mRNA may be degraded during homogenization and incubation in the presence of Triton. If you are interested in isolating mRNA that are still being translated by ribosome, you may purify the ribosomes first and then extract the RNA.
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Re: Effect of triton-X concentration on RNA stability

Postby SyntonicC » Jun 29 2015 4:34 pm

Thanks! I suspect you are probably right.

I was thinking of homogenizing the tissue in the cold room while it is submerged in a layer in of liquid nitrogen right after taking the samples from the -80. I'm going to compare lysates and the PSD fraction for RNA levels to see if I'm losing anything. I at least want a sense of my technique and abilities before I move forward.
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Re: Effect of triton-X concentration on RNA stability

Postby relaxin » Jun 30 2015 10:01 am

You need not work in the cold room. The frozen samples can be stored temporarily on dry ice, you can simply drop them into denaturing lysis buffer and homogenize them immediately with a Polytron homogenizer. The RNA can be purified with commercial RNA spin columns.
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Re: Effect of triton-X concentration on RNA stability

Postby SyntonicC » Aug 14 2015 9:59 am

Just wanted to post an update for anyone else who might come across this thread.

Immediately after dissecting the brain tissue, I flash-froze the samples on dry ice. When I was ready to begin isolating the RNA I worked with the samples on dry ice as they slowly thawed from -80*C. I don't have the homogenizer Relaxin described, only one of those small hand-held ones (and even more basic than one's I've seen online). I just homogenzied the tissue over the next 10 minutes or so. I used RIPA to break open the cell and then isolated the RNA using Norgen RNA isolation columns. From just the hippocampus of a 6-week old rat I received ~290 ng/ul of RNA. This is compared to using an entire left cortex of a rat of the same age before and only getting ~65 ng/ul. So it clearly seems to me that the issue was degradation. I have not yet tried to go through the triton steps yet but I imagine that given the extra time involved (up to an hour) there is sufficient time for the RNA to degrade further. This is the likely cause of my troubles.

I didn't say in the original post but I was isolating the RNA as a QC check for an RNA-IP. The moral of the story is... If your IP isn't working, check the very obvious: Is there enough RNA in your input? I wish I had checked this to begin with instead of troubleshooting the other steps.
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