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miRNA isolation from liquid samples - (Jul/21/2009 )

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I should say I've only ran 8 samples to test if ExoQuick would work. I'm not done testing it out. In general, it causes a greasy pellet to form after the 12hr incubation. From the pellet, I can collet RNA. The RNA content is about equal to that from collecting RNA directly from serum.
There are still a few thing I need to test. I need to see if this is just from exosome or also microparticles; I want to test for exosome markers. I am also still having problems with 260/280 ratio, so I need to try out RNeasy kit with Exoquick and with serum to see how the RNA yield might change.
I've also tried PEG 20000 to cause protein to precipitate from the serum. It seem to also yield about the same amount of RNA.
The 260/280 ratio is beter in ExoQuick than that from serum by far. I get around 1.5 with exoquick compared to 0.8. PEG is around 1.2 260/280 ratio.
My RNA yields are 8-10ng/ul in a 50ul volume.

-Paul Dominguez-

Paul Dominguez on Jun 7 2010, 09:19 PM said:

I should say I've only ran 8 samples to test if ExoQuick would work. I'm not done testing it out. In general, it causes a greasy pellet to form after the 12hr incubation. From the pellet, I can collet RNA. The RNA content is about equal to that from collecting RNA directly from serum.
There are still a few thing I need to test. I need to see if this is just from exosome or also microparticles; I want to test for exosome markers. I am also still having problems with 260/280 ratio, so I need to try out RNeasy kit with Exoquick and with serum to see how the RNA yield might change.
I've also tried PEG 20000 to cause protein to precipitate from the serum. It seem to also yield about the same amount of RNA.
The 260/280 ratio is beter in ExoQuick than that from serum by far. I get around 1.5 with exoquick compared to 0.8. PEG is around 1.2 260/280 ratio.
My RNA yields are 8-10ng/ul in a 50ul volume.


What about a 270 nm peak? that's what makes nanodrop quantitation useless. Did u check if using the same amount of RNA from different samples u get the same Ct for each miRNA?
I'm a little skeptic about exoquick not beacause i've tried it but because it is not yet clear to me what can be called exosome and what microparticle both in terms of size and markers. which markers are you gonna test?

-Fizban-

Paul Dominguez on Jun 7 2010, 09:19 PM said:

I should say I've only ran 8 samples to test if ExoQuick would work. I'm not done testing it out. In general, it causes a greasy pellet to form after the 12hr incubation. From the pellet, I can collet RNA. The RNA content is about equal to that from collecting RNA directly from serum.
There are still a few thing I need to test. I need to see if this is just from exosome or also microparticles; I want to test for exosome markers. I am also still having problems with 260/280 ratio, so I need to try out RNeasy kit with Exoquick and with serum to see how the RNA yield might change.
I've also tried PEG 20000 to cause protein to precipitate from the serum. It seem to also yield about the same amount of RNA.
The 260/280 ratio is beter in ExoQuick than that from serum by far. I get around 1.5 with exoquick compared to 0.8. PEG is around 1.2 260/280 ratio.
My RNA yields are 8-10ng/ul in a 50ul volume.

"From the pellet, I can collect RNA" - just to be sure, could you clarify how you do this? I mean, RNA extraction from the pellet.
and from which volume of serum you get this yield? 400-500ng seems to be not bad, if it is true RNA amount. (How it was determined? could you show the spectrum?)

-comp3v-

Hi,

I am going to try the ExoQuick as well and am very intrested in your discussion !

I get NO RNA at all from NSCLC cell lines (ie. in the culture supernatant) or very few using TRIreagent and with abnormal 260/230 as you guys. I thought using a miRNA specific kit (RNeasy/miRvana) would help but seems like not.
I tried to use equal volumes of TRI/Culture Medium as Chen et. Maybe the miRNAs are too "dilluted"...

None of you did these extractions on cell lines ? If yes what yield did you get ? a lab close to mine uses 1L of culture medium to get 1 g !!!!
Seems weird to me. but the study model is different though.

Waiting for your answers, and to previous questions as well !
I ll try the Exoquick and tell you what I get. Hope to have the sale as you !

cheers.
CP.

-Sero-

Hi again,

Forgot to ask, how much TRIZOL do you guys use to extract from the serum ???

-Sero-

Sero on Jun 14 2010, 12:32 PM said:

Hi again,

Forgot to ask, how much TRIZOL do you guys use to extract from the serum ???


I use 1 mL for each 400 ul of serum. then use 1ml of isopropanol after adding glycogen then proceed as indicated by standard protocol
bye
Fiz

-Fizban-

Forget about nanoDrop or picoDrop its crap for this!

Try to quantify your RNA with Bioanalyser or the Experion RNA (highsense) Chip (even though desalting is needed)! Maybe the concentration is loads lower than you expect!

-Bastard-

Bastard on Jun 24 2010, 04:03 PM said:

Forget about nanoDrop or picoDrop its crap for this!

Try to quantify your RNA with Bioanalyser or the Experion RNA (highsense) Chip (even though desalting is needed)! Maybe the concentration is loads lower than you expect!


Forget Experion too! the only one is bioanalyzer but still it's hard. what i'd like to know is how many people published papers in which they quantitate RNA with nanodrop. beats me ;)
Fiz

-Fizban-

I also trying to extract the miRNA in plasma.
I use the trizol + Qiagen's RNeasy plus kit.
For about 500ul plasma, in 30ul elution, the Nanodrop reading is about 10-15 ng/ul. The 260/280 is alright, but also 230/260 is no gd.
However, it is no problem for the real-time PCR result.

But I would like to ask, this reading is the total RNA but not 100% miRNA, right?
Furthermore, as i know, the circulating miRNA is inside the exosome. Trizol enough to break down the exosome and release the miRNA for purification?

San

-SAN1017-
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