We will shortly be engaged in fieldwork and we need to transport tissue samples (gill, muscle) back to the lab for RNA extraction and cDNA synthesis.
I know RNALater is available for this but we have not been successful with material held even for a couple of days in RNALater and I don't want to risk losing valuable material.
Does anyone know for how long (hours?) tissue samples can be held in Trizol at ambient temperature, if at all? And if we extract into ethanol? Will a few hours at ambient temperature degrade the RNA?
We will not have access to temperatures lower than 0 C (ice) and we will be at least 12 hours in transit.
Ambion's RNAlater FAQ claims 1 day at 37 deg. and 1 week at 25 deg. See: http://www.lifetechnologies.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/rnalater-faqs.html
Trizol will work 3-4 days at room temp. See: http://tools.lifetechnologies.com/content/sfs/productnotes/F_Trizol%20and%20Trizol%20LS-041018-RD-TL-HL0506021.pdf
The key is to homogenize in Trizol or RNAlater, instead of storing the whole tissue or even cutting them into 5mm cubes. RNAlater or Trizol has to physically contact RNAses in order to inactivate them.
I'm sure Trizol will work just fine. Also see this product: http://www.zymoresearch.com/rna/sample-collection-stabilization/dna-rna-shield
Good luck!
Homogenizing might be key if you can do it. I use both TRIzol and RNALater for stabilization of viral RNA in rodent spleen in the field in West Africa. Cold chain for us is ice and a cooler and sometimes we are out for a couple days in the heat. RNALater is designed to penetrate tissue and the techs I've talked to say that it's better to keep tissue at RT for a few hours to allow the solution to penetrate tissue before freezing.
I've had success with TRIzol as well, but keep in mind spleen is really soft tissue. I've thought about homogenizing but can't because of biosafety issues. Have had some success with Whatman FTA paper but this was using whole blood.
I have had success storing the samples with RNA later in a cooler (we did not had ice either but just used icepacks and a fabric cooler) for the whole day in the field and then keeping them at 4C for up to 7 days before dissecting the tissues. Here I attach reference of the paper we describe the methods. If you are interested and want more details just contact me.
Article Expression profiles of metamorphosis-related genes during na...
If you are collecting gill and muscle and you have small clean place for cutting this material into 2mm pieces by blade , these pieces you can directly put it into Tryzol and vertex and keep it on ice till you come to lab. than you keep this tube in -20 freezer till further analysis. I think it will work for you. Try the same.
One more suggestion, if you are using RNA later and not getting good result, you can discussed this problem with manufacturer. they may suggest you better way to do.
DR. PRAVIN D. POTDAR
Andrei might be correct, but the key is that you HAVE TO SOLUBILIZE it. If you can bring a polytron type device and trizol, it *might* be OK I actually am not certain about RT storage of the homogenate becasue the reducing agent might go off. Try it, homogenize a sample in TRI, let it sit a few days, run it on a gel. If you can break the phases and alcohol precipitate it, I'd leave it as an alcohol and salt precip. Thats pretty stable as well.
BTW, I inventer RNAlater. If your tissue samples are small enough it really should work. I assume fish and mouse tissues are similar. Mouse tissues were OK at 25C for a week (based on Northerns, not NGS). I assume if you dice the thing up in RNAlater it should be fine. Cant imagine it wouldnt be.
Eric
Oh sorry, one more thing, if you actually isolate the RNA, the RNA/DNA shield stuff might work, but so would low pH TE. I still like the ethanol precip or RNAlater.
A glass homogenizer would work to enable grinding the tissue to allow the trizol to protect your samples. You can also transfer your samples in dry ice to which you added some alcohol. This should keep your samples cool enough for few hours. homogenized samples in trizol could survive in -20C for years. Also RNA is safe as ethanol-salt precipitate for years in -20C.
yes, you will need a glass homogenizer for a tissue to grind. The trizol will protect your RNA sample as it helps in its stability. You can directly add tissue pieces to trizol, vortex and let it cool and so on....
Way don't you tray to homogenize your tissue when you colect them, with a scisor to chop, and them with a tissue grinder inside the eppendorf of 2ml? If the tissue is soft you can tray to homogenize with a seringue and a needle also, passingit throuugh it.
I sucede doing the fírst sugestion with mice foot paw.
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