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Subject: RNALater for seaweed samples - compiled responses
From: Erica B Young <[log in to unmask]>
Reply-To:Erica B Young <[log in to unmask]>
Date:Tue, 22 Nov 2011 10:13:59 -0600
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Many thanks to those who responded to my query about using RNAlater to preserve seaweed samples. Below is a compilation of the responses. If anyone has further thoughts, please respond to the list. 
The 'gist' of the responses seems that yes, RNAlater should work but key recommendations are to cut the thallus up in small/thin pieces, if possible let it incubate at 4oC before freezing in RNAlater (to allow percolation into the tissue), and keep cool as possible during shipping. Also remove as much water as possible from outside of thallus and clean to remove epiphytes/bacteria before transferring to RNAlater.

Thanks again for sharing your experience.

Erica

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First, I used RNAlater several  times for customers while I was at the CCMP.  We found the best results were when we ground the cells in the RNAlater using a hand tissue  grinder.  Bob Anderson
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We have primarily used it with microalgae, but at least some of our experiences should translate to macroalgae.  RNAlater does not include a permeablizing agent, and in our experience we get much better preservation if we freeze the sample first, then transfer it into RNAlater.  I think this is probably because it allows the RNAlater to rapidly penetrate the cells.  I would suggest that you try dipping the thallus in liquid nitrogen (or laying it on dry ice) and then dropping the frozen tissue into (liquid) RNAlater.  Of course grinding the tissue in RNAlater would have a similar effect, but it would be more labor intensive.   Charles Delwiche
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I don't have experience with macroalgae, but I used RNALater to preserve several zooplankton samples from the Kimberley. I love RNALater (but not its price), and it worked perfectly to me. I don't know how the plant cell characteristics may affect the preservation, so if you should talk with someone who tried to use RNALater with plants.
Here are some general tips that may help you:
- RNALater needs to penetrate deep in the tissue in order to preserve the RNA, so the piece of material to be preserved need to be relatively small. For zooplankton, I  obviously did not have to worry about that. I don't remember exactly, but I think the piece of tissue must not have more than 1cm in any side. I believe plant tissue may be more difficult to preserve than animal tissue. So, if I were you, I would cut the thalli in small pieces to preserve.
- Remove as much saltwater from the as possible before preserving in RNALater. I used to blot dry the samples before preservation. RNALater is a very saline solution that will not freeze at -20°C, but if there is saltwater, it may freeze.
- I had a big headache with gelatinous material in my samples, such as jellies, blobs of mucus, and other weird things. It looks like RNALater does not penetrave very well in gelatinous material, so all samples I had jellies, for instance, the gelatinous material froze at -20°C. I don't know if the RNA of jellies was preserved, but it looks like the rest of the sample was preserved ok. If the macroalgae you are preserving has a lot of mucus or secretions around it, I would try to scrape it off before cutting the tissue and putting it in RNAlater.
- Respect the ratio tissue volume to RNALater volume. I had success using a little bit less RNALater than the volume indicated in the protocol that comes with the product, but all my organisms are really tiny. I used 8ml of RNALater to 1ml of mixed zooplankton. Considering the cost of RNALater, when you see more than half of the RNALater in the tube not in touch with the sample, it looks like you are throwing money away, but you should not reduce the amount of RNALater.
- You should keep the sample for at least 24hours in the fridge (4°C) before transferring it to the freezer. This period is very important to allow the RNALater to penetrate the tissue. I don't know about plant tissue, but I believe you would need to keep the sample in the fridge for two days before freezing.
- After 1-2 days, remove the samples from the fridge and transfer to the -20°C freezer for long term storage. The RNALater should be liquid at -20°C. I keep my samples at -40°C and most RNALater in the tubes is still liquid, but very dense.
Regarding transporting: as far as I remember, the protocol says that RNA from samples preserved with RNALater may be ok for up to 1 or 2 days under room temperature, but I wouldn't risk losing my samples. If you keep the samples could, you will be ok. I used a dry shipper to transfer my samples (Qantas was ok with that). A cheaper alternative is dry ice, but I don't know how airlines deal with that.
RNALater is not toxic, so I think customs may not have a problem with that - check with them. Just in case, send with the samples RNALater MSDS and Qiagen brochure about RNALater.
I am copying this message to Pia Winberg from the University of Wollongong as this information might be interesting for her as well.
Felipe Gusmao
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Yes we have used RNA successfully with macroalgae but I am forwarding to my genetics expert collegue Lisa Kirkendale at our lab. Lisa has used this mostly with Ulva and we have used it successfully for long term storage with Porphyra. Lisa is better on the details with browns.  Pia Winberg
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 We have used RNAlater extensively with red algal tissue. With thick browns I think you would need to slice the thallus into thin strips to make sure you had good preservation. Make sure to clean the surface as much as you can, we sequenced half the ocean the first time we tried to get ESTs from our reds.
-Chris E. Lane
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I am afraid we haven't tried RNALater on Fucus and Laminaria sporophytes, but we have successfully been using it on Ectocarpus and Laminaria gametophytes. 
It is possible to extract both DNA and RNAs from such samples. We simply pellet the biomass, remove the RNALater, and start with the extraction protocol as with fresh material. We tend to keep samples frozen as much as we can in the lab, but it is OK to ship samples in RNALater in the post.
Sorry I have no idea about US custom regulations, but cannot anticipate too many difficulties: all samples will  be dead and RNALater is non toxic.
Last point: RNALater is now available from Sigma at a much lower price than from Ambion. Claire Gachon
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Hello erica, i has experience in use RNAlater in fish tissue samples for more than 4 days, and have no problems with It. At 4ºC is best to the shipping.  Claudio Jofre
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Erica B. Young        Associate Professor
Dept Biological Sciences and School Freshwater Sciences
Univ Wisconsin-Milwaukee, WI 53211
Ph.  414 229-3257     http://www.uwm.edu/~ebyoung/index.html
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