PAXGENE Blood RNA tubes, Plasma RNA extraction?

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PAXGENE Blood RNA tubes, Plasma RNA extraction?

Postby Aidanmc » Feb 01 2009 8:03 pm

Hi All,

So I have blood collected in PAXGENE blood RNA tubes and I want to extract RNA from the plasma of this blood. The PAXGENE blood RNA extraction kit is for extracting INTRACELLULAR RNA from whole blood collected in PAXGENE tubes. Does anyone know if i could spin the tubes I have and extract RNA from the supernatant produced (which will contain plasma diluted in the RNA preservative solution found in paxgene tubes) using TRIZOL?

any help would be appreciated,

Thanks
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Postby relaxin » Feb 02 2009 10:56 am

It seems to be "doable". But since you have a liquid sample, you may need Tri-Reagent-LS (or Trizol LS) instead of regular Trizol.
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Postby Aidanmc » Feb 02 2009 6:41 pm

ok thanks, do you think the RNA preservative in PAXGENE tubes (i am guessing similar to RNAlater: 25mM Sodium citirate 10mM EDTA, 10M ammonium sulphate) will hinder the Trizol extraction?
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Postby relaxin » Feb 03 2009 9:19 am

It should not hiner the Trizol extraction.
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Postby affenur1 » Feb 03 2009 10:19 am

Hi Aidanmic,
I suggest, make 350ul aliquots from the supernatant put 1ml trizol together mix well and process further!
so long
Urs
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Postby Aidanmc » Feb 09 2009 7:58 pm

thanks affenur1!! have you tried this method yourself?
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Postby Aidanmc » Mar 11 2009 1:10 am

I tried this method as follows:

525ul of PAXgne supernatant with 1125ul of TRIzol LS,

yeilds ~1.5ug nucleic acid (nanodrop conc) which is good, however the RNA is not clean! 260/280 ~1.7 and 260/230 ~0.3 (there is a large peak at 230 which i reckon is guanidine/phenol). Also the peak is not at 260nm but instead closer to 270, why is this?

any suggestions is appreciated!

thanks!
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Re: re

Postby mchlbrmn » Mar 11 2009 1:23 pm

Aidanmc wrote:I tried this method as follows:

525ul of PAXgne supernatant with 1125ul of TRIzol LS,

yeilds ~1.5ug nucleic acid (nanodrop conc) which is good, however the RNA is not clean! 260/280 ~1.7 and 260/230 ~0.3 (there is a large peak at 230 which i reckon is guanidine/phenol). Also the peak is not at 260nm but instead closer to 270, why is this?

any suggestions is appreciated!

thanks!
Phenol has an absorption max at A270.

I wonder if the cells could be pelleted/concentrated in the RNA preservative, before RNA extraction? Maybe the preservatives protocol says (I think RNA later protocols discuss this).
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Postby Aidanmc » Mar 12 2009 11:19 pm

I am trying to get at the "cell Free" RNA as this supernatant that I am extracting from is the result of a primary spin of whole blood collected in PAXgene RNA tubes.

The Pellet containing the cells I will also extract from but I want to see if there is extractable RNA left in the supernatnant

Any suggestion on how I could clean up the phenol contamination and whatever is causing the low 260/230?
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Re: PAXGENE Blood RNA tubes, Plasma RNA extraction?

Postby Ingo » Apr 26 2009 8:04 pm

To clean up the RNA one can precipitate the RNA again. Trizol extracted RNA often has salts and or phenolic contamination if one is not careful when removing the aquous layer.
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Re: PAXGENE Blood RNA tubes, Plasma RNA extraction?

Postby JoGo » May 06 2009 12:35 pm

Aidanmc,
Did you have any luck getting better RNA? I am having the exact problem that you are. I have been using 0.25 ml of blood/PAXgene solution with TRIzol LS to try to get total RNA from the blood samples and I am ending up with a weird looking white pellet at the end that doesn’t have the same properties as nucleic acid. I have tried dissolving this in H2O and re-TRIzol extracting, but I am ending up with the same weird 260/280/230 readings. Any advice you have would be great.
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Re: PAXGENE Blood RNA tubes, Plasma RNA extraction?

Postby relaxin » May 06 2009 2:08 pm

Ingo wrote:To clean up the RNA one can precipitate the RNA again. Trizol extracted RNA often has salts and or phenolic contamination if one is not careful when removing the aquous layer.


Repeated ethanol precipitation will not remove phenol. You need to extract the RNA solution with chloroform prior to ethanol precipitation.

Most people clean up RNA isolated from Trizol procedure with Qiagen RNeasy spin column. This will remove both salt and phenol.
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Re: PAXGENE Blood RNA tubes, Plasma RNA extraction?

Postby Suzanne » May 06 2009 2:55 pm

JoGo wrote:Aidanmc,
Did you have any luck getting better RNA? I am having the exact problem that you are. I have been using 0.25 ml of blood/PAXgene solution with TRIzol LS to try to get total RNA from the blood samples and I am ending up with a weird looking white pellet at the end that doesn’t have the same properties as nucleic acid. I have tried dissolving this in H2O and re-TRIzol extracting, but I am ending up with the same weird 260/280/230 readings. Any advice you have would be great.


Try pelleting the material in the paxgene/blood mix first and start with the pellet. Normally the pellet is washed to remove the preservative. You can try extracting the pellet with TRIzol without washing it and if it still doesn't extract, try washing the pellet with some RNase-free buffer, re-pellet, and extract that.
You won't have too much RNA from 0.25 ml. Is it human blood?

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Re: PAXGENE Blood RNA tubes, Plasma RNA extraction?

Postby JoGo » May 06 2009 4:27 pm

Suzanne wrote:
JoGo wrote:Aidanmc,
Did you have any luck getting better RNA? I am having the exact problem that you are. I have been using 0.25 ml of blood/PAXgene solution with TRIzol LS to try to get total RNA from the blood samples and I am ending up with a weird looking white pellet at the end that doesn’t have the same properties as nucleic acid. I have tried dissolving this in H2O and re-TRIzol extracting, but I am ending up with the same weird 260/280/230 readings. Any advice you have would be great.


Try pelleting the material in the paxgene/blood mix first and start with the pellet. Normally the pellet is washed to remove the preservative. You can try extracting the pellet with TRIzol without washing it and if it still doesn't extract, try washing the pellet with some RNase-free buffer, re-pellet, and extract that.
You won't have too much RNA from 0.25 ml. Is it human blood?

Best,
Suzanne


Suzanne,
Yes it is human blood. I am interested in obtaining total RNA (including microRNAs) from the blood, not just from cells, so I don't think that pelleting at the beginning is really an option for me. I actually have been performing the extractions from multiple 0.25 ml samples and combining the final products. I think my main problem is coming from whatever salts/etc that are present in the PAXgene tubes when I go to precipitate the RNA with isopropanol. I think I might put the aqueous layer straight onto a spin column instead of precipitating. Thoughts???
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Re: PAXGENE Blood RNA tubes, Plasma RNA extraction?

Postby Suzanne » May 06 2009 6:22 pm

I think there might be some references using PAXgene for miRNA work- have you checked?
Usually, to purify the small RNA on a spin filter column, you just need to increase the amount of ethanol used in binding.

The very first step of the paxgene protocol is to pellet the cells/debris. I would speculate that the total RNA is not free yet. If it were, the protocol wouldn't work. So the miRNA and total RNA are still safe inside of cells and pelleting the sample should not be a problem.

The next step after pelleting and washing the pellet is to lyse in a mixture of proteinase K and chaotropic salts. That is when the cells are lysed and the RNA and DNA is released. In your case, you can lyse the pellet with TRIzol.

I would give it a try and see what you get. It's worth a try.

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