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Trizol Extraction of PBMCs, low 260/230 - (Nov/18/2010 )

Hello,

I'm new to this board and have been reading it for a long time, and I think you are doing a really good job.I decided to register myself, because I have problems regarding my RNA Isolation. I want to isolate RNA from cultured PBMCs (round about 10^6 or less cells).
When I started up with Trizol I had very bad 260/280 ratios (~1,3) but I got along with that and reached a point where it is between 1,7-2.0.
But now I'm faced with bad 260/230 ratios (between 0,1 and 0,7).
I did the isolation according to the TRIpure protocol.
I put the extinction curve from the nanodrop reader into the attachment, which consists of two isolations.
In the first Isolations I had much excess ethanol in my samples (15-20 microliters) which might cause the bad ratios.
Now I tried to optimize the progress in the second RNA isolation and discarded all supernatants always keeping sure that the pellet is in the eppi. I also could see the dried transparent pellet after the last ethanol washing step (75%).I diluted the pellet in 25 microliters nuclease free water and incubated it for 10 mins at 60°C and stored it at -80°C.Today I went to measure it in the nanodrop. I could see that there was almost none RNA left. I had a yield of 30ng/per microliter, normally I had values like 300-1000ng per microliter. Though the 230nm extinction was less regarding the other isolations, I have a very bad 260/230 ratio, because there is almost no extinction at 260nm.
Does anybody know why I'm having such bad ratios?Troubleshooting the second isolation: Don't the curves show that my contamination got less and that I somehow just lost my pellet? The pellet was at first slight whitish and after drying it got quite transparent and filamentous. If it wasn't RNA in my pellet, because the nanodrop doesn't show much extinction at 260nm, what was is then, regarding that the 230nm and 280nm extinction is much lower than in the first isolation.
Could it be, that the RNA just did not dilute in the nuclease free water, though i also heated it for 10 mins? I used four samples for the second isolation. I overdried the first sample for along 40 mins. I dried the last pellet for about 10 mins and there is no difference in results...

I'm getting quite frustrated with Trizol... I would be thankful for every advice,

best regards Alex
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-AlexanderA-

Hello AlexanderA,

welcome to Bioforum :)

these notes are from my experience using TRIzol :

i used to use TRIzol, with excellent results in extracting RNA from Peripheral blood & bone marrow samples.
though, i have NEVER dried my samples at 60 C ...
i always did it at room temperature for 10 minutes ...
i feared loosing my RNA ...

i used to measure the white blood cells before undergoing the extraction procedure,
and then upon the size of the seen pellet along with the CBC i decided the amount of ultra pure water added,
so it varied between the samples ...
in times it was down to 15 ul, others high to 45ul ...

hope my notes will benefit you ...

-nightingale-

Hi thanks for answering,

the reheating to 60C wasn't meant for drying because I already redissolved the pellet in Nucelease free water.
I did that according to the TRipure protocol to dilute the RNA properly.
Could you give me any information about the critical steps regarding the RNA Isolation? Do you completely aspirate all the supernatants? How much supernatant is still tolerable during the isolation process?
Which colour should the pellet have after the last ethanol washing step and after having let it dry for approx. 10 mins?
Which 260/230 ratio is tolerable for downstream applications like RT-PCR?
thanks again! Alex

-AlexanderA-

you are welcome AlexanderA :)

the reheating to 60C wasn't meant for drying because I already redissolved the pellet in Nucelease free water.


okay ... forgive me for missing this !
i remember such protocols suggested like this step ...
forgive me ...

regarding good step on RNA isolation, kindly see the link below :
it is from Ambion RNA's site ...
Practical Tips for Handling RNA

i believe from the critical steps is gentle vortexing of the pellet, after dissolving it with the nuclease free water.
and letting the pellet completely dry from EtOH.

but, here you have this site ... explore it :)

Do you completely aspirate all the supernatants? How much supernatant is still tolerable during the isolation process?


after which step ???
do you mean the EtOH ??

Which colour should the pellet have after the last ethanol washing step and after having let it dry for approx. 10 mins?


white coloured pellet ...


Which 260/230 ratio is tolerable for downstream applications like RT-PCR?


1.8 - around 2.0

Organic contaminants like phenol and other aromatic compounds, TRIzol, and some reagents used in RNA extraction absorb light of a 230 nm wavelength. Samples with a low 260/230 (below about 1.8) have a significant presence of these organic contaminants that may interfere with other downstream processes like RT-PCR

--- taken from organic contaminants

-nightingale-

please del

-Rashtagul-

Hi nightingale,

thanks again, the Ambion's tips seem very useful to me! :)
I'll have another try on Monday and I'll tell you about the results

Bye Alex

-AlexanderA-