Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Ethanol contamination in RNA - (Jul/24/2012 )

Pages: 1 2 Next

hi, i'm new in doing RNA works. the method i used to extract RNA is using Trizol. this is my 2nd trial and i still got problem to completely dry my RNA. i left them in BSC for almost 2 hours for drying.after measure it with Nanodrop, there's still ethanol contamination in it.i've been told that ethanol is an inhibitor so i need to get rid of it. so, i would like to know how problematic is this ethanol if i'm going to proceed for Real-Time. the method i use is Taqman and the machine is ABI 7500 fast system. thank you

-pLASMID-

First of all, Trizol extraction is not suitable for Real-Time PCR. It is true that it might work, but it is strongly recommended to use RNA extraction kits, e.g. RNeasy from Qiagen or any other brand.

About the Ethanol issue, we normally add 70% ethanol in the last step of extraction, air dry for only 10 min and then add 30 ul of water with 1 ul of RNase inhibitor. Drying more than that might harm your RNA. At the air dry step you need to invert your tube and use a micropipette tip to remove ethanol. An empty tip sucks in excess ethanol from the tube. Do this quickly and make sure ethanol is removed, then air dry. Don't touch the pellet, although RNA pellet is usually difficult to see. If the concentration is too high then you must see a transparent pellet.

I personally have had no problem with ethanol contamination. I also don't think the contamination you see with nanodrop is really because of ethanol. It could be protein contamination?

-Curtis-

thank u..i have higher peak at 230.i did RNA clean-up and the result is not good for some of my RNA. the yield also very low. i'm very frustrated now.maybe my techniques is not good. or i'm doing it wrong way. i think i'm going to start over again.

-pLASMID-

ethanol is a temporary inhibitor of enzyme.
after adding 70% ethanol, it is better to centrifuge the vials for 1 min with speed of 10000 rpm and more, then remove alcohol by pouring out or by pippet cerfully. during pouring out, see the pellet always. then you can see a little alcohol at the end of the vials. remove them by long tips as much as you can.
now put vials in Hotblock at 37 degree centigrade for 15 to 30 min and take them when there is only a drop, for exaple 1 mm in diametter or less. do not worry if they have been compelety dried.
now add 20 to 50 ul of clean water and vortex for a second and put it on ice for 5min and then use it or keep it in -80 or less.

-----
Babak

-memari-

We have no ethanol contamination with Trizol RNA isolation. Are you sure it's ethanol? Perhaps if you seen residues on the vial then you are.. 70% ethanol doesn't evaporate very avidly at RT.


You can try heatblock method, but the best way is careful removal of all drops. I don't prefer pouring it out because that causes drops on the sides, but first putting the vial in the centrufuge in a way you know where your pellet is even if it's not visible (usualy opening facing down, the pellet would be on the oposite side near the bottom, but not at the very bottom). Then you take a 1000 pipette and slowly but continuously aspire the ethanol, keeping the tip at the side oposite to pelet, you can even touch the very bottom, because pellet isn't there, but don't distrub it and if it's visible check all the time that it's not not sliding down. Just do it slowly enough that the liquid on the sides manages to slide down as you aspire, otherwise it will leave drops.Take whatever is possible with 1000 and carefully take it out not touching the sides with the tip. Generaly you don't need to push it hard to remove completely everything at this step, because 1000 tip is too robust anyway. Then you take 200 or smaller volume pipette and new tip, the narrower the tip, the better, but it must be bigger volume than what is now left in vial. Now you keep the vial upright, keep on the side oposite of pellet and touch the bottom with a new tip (remember the pellet is not there if you didn't disturbed it or slided down) and get out the rest, again not touch the sides.


If the pellet slides down, you can sometimes stick the tip just beside and slooowly drain it, but with that you must be extremely careful and that is more advanced.
If you manage to get drops on the sides inspite all this, use the most narrowest tip to drag it up and there absorb with capillary efect.
If you have at least commonly low-retention plastics, that should eliminate all visible ethanol, then you leave it to dry in the hood 10 minutes maximum.


Curtis: What reasons do you have for claiming Trizol is not suitable for real-time? It's been proved that basic Trizol isolates are troublesome for microarrays and such, but these are aplications extremely sensitive for RNA purity. Trizol RNA also has a problem with DNA contamination, but that is simply solved by DNA-free kits. Undesirable Trizol caryover contamination can be prevented by the right handling of the phase separation.

-Trof-

you named the issues already. For first timers it is not recommended. If you are professional then you can use Trizol if you want. The yield is even higher.

-Curtis-

I see, it's easier and more robust to get good quality RNA for real-time with column kits and on-column DNAse treatment. I agree.
However the yield is not only lower but more importantly limited in the column method, we use mainly human blood in amounts sometimes 6 times exceeding the column limit. Also storage in the form of Trizol lysate is more stable than storage of purified RNA. This is also very important reason for us. There is also evidence, that column isolation gets only certain NA lengths (not too small, not too long) than organic lysis, though that is not relevant unless you for require intact long transcripts for RACE methods or so.

-Trof-

i'll try to be careful next time. do i need to rinse the cells with cold PBS after remove all media before adding trizol? or is it optional step?

-pLASMID-

pLASMID: Yes, do a PBS wash, it cleans the cells from the remnants of medium.

-Trof-

can RNA be cloned to get more?

-pLASMID-
Pages: 1 2 Next