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Can't get clean RNA prep with TRIzol


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#1 wilk0x

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Posted 08 April 2010 - 07:14 PM

Hi,

I'm trying to extract RNA from cells frozen on a polycarbonate filter, using TRIzol. The extraction seems to go exactly as expected, until I run the final product on the nanodrop, and get massive peaks at ~225 and 270, indicative of TRIzol contamination (the 260/230 is always <0.5). I've tried repeating the chloroform cleaning step and the ethanol wash step, re-precipitating with isopropanol, and using phase-lock heavy tubes to separate the phases but nothing seems to work. The most recent protocol I tried is pasted below. Can anyone offer advice?

Thanks in advance



-Incubate filter at 40 degrees until thawed completely
-Fine chop filter
-Place filter strips in a 50 mL Falcon tube
-Add 4 mL TRIzol
-Vortex 1 minute
-Incubate RT 10 minutes
-Vortex 1 minute
-Centrifuge 10 mins at 5000 RPM at 4 degrees
-Pipette supernatant into 4x Eppendorf tubes
-Add 200 uL chloroform to each
-Vortex 10 sec
-Incubate RT 1 min
-Centrifuge 13000 RPM 10 mins at 4 degrees
-Transfer upper phase to 4x pre-spun phase-lock heavy tubes
-Add 200 uL chloroform
-Vortex 10 sec
-Incubate RT 1 min
-Centrifuge 13000 RPM 10 mins at 4 degrees
-Transfer upper phase to 4x fresh Eppendorf tubes
-Add 500 uL isopropanol
-Incubate at RT 10 mins
-Centrifuge 13000 RPM 5 minutes
-Remove supernatant
-Add 50 uL 70% ethanol
-Vortex 10 sec
-Centrifuge 13000 RPM 5 minutes
-Remove supernatant
-Add 50 uL 70% ethanol
-Vortex 10 sec
-Centrifuge 13000 RPM 5 minutes
-Remove supernatant
-Air dry 10 mins (don't overdry)
-Resuspend 30 uL DEPC water at 40 degrees for 15 mins

#2 Curtis

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Posted 08 April 2010 - 07:43 PM

I normally use Trizol to extract RNA from virus samples or to extract total RNA from Eukaryotic cells and it works really fine, even better than Qiagen kits. Even the purity is higher.

my suggestions:

1- if you are adding 30 ul water, then you can add 1 ul of RNase inhibitor in the end.

2- Nanodrop is like that. Sometimes it makes fun. However it depends for what application you want your RNA for? do you want to do RT-PCR...then don't worry, go ahead with the experiment.

3-why these cells are frozen on polycarbonate filter? I never heard of this before sorry....carry out each step on ice,...except the isopropanol addition step. this step needs to be at RT, incubate.....centrifuge for 10 min because 5 min might not be enough

4- instead of vortexing with EtOH for 10 sec, just pipette up and down with DNase-RNase free tips, as many times as you want....sometimes the pellet remains as a thick precipitate. you needs to dissolve this to clean it, ain't that right? otherwise what's the point of adding EtOH for the washing step?

5- is your precipitate white, or transparent?.....better be transparent.

#3 wilk0x

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Posted 08 April 2010 - 08:01 PM

Thanks for your reply Curtis.

2- Nanodrop is like that. Sometimes it makes fun. However it depends for what application you want your RNA for? do you want to do RT-PCR...then don't worry, go ahead with the experiment.


I'm preparing the RNA for sequencing so it needs to be very pure. Also with the big 270 peak swamping the 260 RNA peak it's impossible to tell how much RNA I actually have!

4- instead of vortexing with EtOH for 10 sec, just pipette up and down with DNase-RNase free tips, as many times as you want....sometimes the pellet remains as a thick precipitate. you needs to dissolve this to clean it, ain't that right? otherwise what's the point of adding EtOH for the washing step?


Good idea, I'll try that.

5- is your precipitate white, or transparent?.....better be transparent.


Because the filters have varying biomass (some have lots, some hardly any) I've seen the full range of sizes and colours of pellet, from invisible to huge, and from transparent to white to brownish. It never seems to have any effect on the purity.




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