Rat Brain Tissue RNA Isolation with TRIzol - (Mar/20/2007 )

Hello,

I have been attempting to isolate RNA using the TRIzol Plus kit. The core that analyzes my samples uses an Agilent Bioanalyzer, and reports that my RNA samples are degraded. I've managed to pinpoint some issues with sterility and temperature, and plan to use RNAase ZAP wipes from Ambion & perform the TRIzol procedure on ice for future trials. It has also been suggested that I use liquid nitrogen to flash freeze the tissue. I have questions regarding the length of time for tissue harvesting. The tissue I am using is rat brain, specifically the hippocampal region. The person who does the brain harvesting is able to remove the brain & dissect out the region of interest in approx. 5-6 minutes; does anyone know if this is a reasonable length of time? It is performed on a tray chilled over ice and then immediately transferred to a tube in dry ice, but is it likely that the length of time it takes to dissect out the hippocampus is contributing to the degradation? Also, if I use the liquid nitrogen, should I pour it directly over the tissue, or should I set the tube containing the tissue in a container of liquid nitrogen? Is it possible to get good results without using liquid nitrogen and freezing on dry ice instead? Thanks to anyone willing to answer even some of my many questions; as I'm sure you can tell, I am very knew to this type of procedure!

Hi usclab123,
I think the time of brain harvesting is not too long. When you get the hippocampal region , maybe you could immediately keep it in -80 centi-degree for a shout time, or shear it to small pieces in grind at the same time pouring liquid nitrogen into it and grind it down. liquid nitrogen should add any time it is evaporation to dryness.
Then you can translat them to a container, isolate RNA under the protocol of the TAIzol Plus kit.

Hi,

My experience is with fat tissue not brain but they have a lot in common as both contain a lot of lipids. The 5-6 minutes isolation shouldn't be too much of a problem. Its how you freeze it and how you treat it after freezing that is critical. We isolate the fat then wrap it in silver foil and drop the silver foil packet directly into liquid nitrogen - dry ice won't freeze the tissue quick enough. The frozen samples are then transfered to -80 THEY MUST NOT BE ALLOWED TO THAW OR WARM UP AT ALL AT ANYPOINT THAT ALSO MEANS DONT HANDLE THE PACKETS WITH HANDS. An alternative to this is homogenize the fresh tissue directly after isolation in Trizol - the homogenized tissue is now stable. When you want to prep the tissue it is very important not to let it thaw - transport the tissue from freezer either in liquid nitrogen or burried in dry ice then either grind up the tissue in a pestle and mortar under liquid nitrogen or very rapidly addd the frozen tissue to a ball mill homogenizer tube and homogenize straight away. I must stress any ammount of thawing of the tissue before the trizol penetrates will result in some RNA degredation. lastly after homogenizing do you do an initial spin in trizol to remove lipids???.

Hope this helps

Matt

Hi,

Thank you all for your replies!

Matt--Thanks for all of your tips, that really helps a lot. No, I have not tried doing an initial spin in Trizol. The protocol that I have (the one that came with the packaging from Invitrogen) said to add TRIzol, incubate at room temp for 5 minutes, then add chloroform, incubate for 2-3 min, and then centrifuge. So I had not previously been centrifuging prior to that step, but if you think it will help I am more than willing to try How long should I centrifuge for after adding TRIzol, and do I still incubate for 5 minutes? Thanks!

Reagent--That is actually something we have been discussing. Some of the people involved were in favor of it, but then others seemed to think it wouldn't make that big of a difference. So I've mostly just been trying to gather as much information as possible to present to everyone so we can hopefully make a good decision on the best way to proceed. But it's definitely on the list of potential things to try. Thanks for the input

Thanks!

Hi usclab123,

do the initial spin only if you don't want to copurify DNA!

Krümel

Yes the initial spin will pellet large Mw DNA but it also helps reduce any lipid contamination as lipids will form a layer on the surface of the Trizol after this spin at 4c. Lipids are a problem for anyone trying to isolate RNA from fatty tissue as a small ammount of lipid will cause carryover of phenol into the aqueous phase. Although brain is classed as a fatty tissue, I suspect in your case as I would imagine the tissue is small there will be virtually no lipid visible and therefore the initial spin may not be necessary.

Matt

Thanks Krumel & Matt!

I'm not worried about copurifying DNA, so that won't be an issue, but mucho thanks for pointing it out I may try running two separate samples, one with spin and one without to see if there is a difference in the final outcome. Thanks; have a great weekend!

Usclab123

Hi again,

I have one more question that is probably a rather silly one, but here goes! I have some tubes that I am considering using for the procedure that say they were sterilized using gamma radiation. The pipette tips I have been using specified that they are guaranteed RNAse free, so I'm worried since these tubes don't also specify that. Gamma radiation sounds to me like it would make the tubes sterile, but I've also heard that RNAses are tough little guys, so does anyone know if these tubes are ok for me to use (ie, if they will be RNAse free)? Thanks so much!

usclab123