Hello!
In my ongoing project I am trying to isolate the RNA for transcriptomic analysis from a giant sulfur bacterium, Achromatium oxaliferum (kindly see the project description for more details).
We have tried a couple of buffers now and have run into a few issues.
Firstly, it is important to mention that since the bacterium has not yet been cultivated, we are dealing not with pure cell cultures, but with environmental samples. We are taking out ca. 1 L of sediment sample, as the bacteria of interest resides on the oxic/anoxic border of the lake sediment. Therefore, since it is crucial to "freeze" the expression pattern of the bacteria's natural state, we have to fix the whole sample at once, before fishing the cells out of the sample and performing cleaning procedures on them.
For this reason, Formaldehyde-based fixatives can not be employed, as FA is highly toxic and it is not possible to work safely with the large amount of the chemical required for this procedure.
First attempt was to employ the RNAlater buffer. However, its high salinity resulted in the cells floating, instead of sinking. Sinking cells are available for hand-picking under a binocular, which has been the technique for studying these giant bacteria for decades. However, floating cells can not be seen and thus collected.
Another buffer that we tried is a zinc-based buffer. In this case, the chemical composition of the buffer results in the bacterial EPC to become highly sticky - or the cells start producing increased amounts of EPC as a stress response, we are not really sure - making it again impossible to isolate and clean the cells manually, like we normally do.
Additionally, the cells can only be seen and isolated due to the fact that they bear large calcium carbonate solid inclusions, which are shiny under the binocular light, which allows for hand-picking. Low pH of both buffers dissolve these solid inclusions, rendering cells invisible and thus not possible to isolate.
Given all these empirical considerations, we are now in search of a buffer that:
a) is not toxic
b) has a pH of at least 7+
c) is not highly saline (liquid enough for the cells to not float)
d) does not render the EPC of bacteria increasingly sticky
e) preserves cell integrity
Any input, thoughts suggestions and considerations are very welcome!
Thank you for your time!
Artur Zaduryan
I do not have expertise with the volumes you are using or fixing bacteria, however, there is a reversible RNA crosslinking fixation compund called DSP (dithiobis succinimidyl propionate) which works very well with human cells and tissues. perhaps you could look into this?
I also don't know about volumes or bacteria, but we have had very good results using modified methacarn (MMC) as a fixative for tissues from which we needed to harvest high quality RNA. MMC can be made in the lab from methanol and glacial acetic acid (8:1 v/v).
Thank you for answers!
Unfortunately acetic acid would disrupt the cells, and we need to fish them out after we fix them, so that doesn't work for us
If I remember correctly, glacial acetic acid is added to the methanol in MMC in order to minimize tissue contraction, and I think it also penetrates tissue quickly.
In your case, you won't need to worry about either of those issues, so maybe going with just methanol itself will work for you?
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