Searching for the right RNA fixative

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Searching for the right RNA fixative

Postby ArtieFinnigan » Feb 07 2018 5:36 am

Hello all!

In my ongoing bachelor project I am trying to isolate the RNA for transcriptomic analysis from a community of giant sulfur bacterium, Achromatium oxaliferum (kindly see the project description for more details).

We have tried a couple of FIXATION 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 in the lake sediment. Therefore, and 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 routinely 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 well and thus collected in a time-efficient manner.

Another buffer that we tried is a zinc-based buffer. In this case, cells (the EPC) stick-aggregate together and with all the organic matter, . Is it because of the bare chemistry of the buffer, or because cells produce increased amounts of EPC as a stress response - we do not know.

Additionally, the cells can only be seen and isolated due to the fact that they bear large calcium carbonate solid inclusions, which appear as visible shiny crystals under the binocular light, which allows for hand-picking. Due to the low pH of both buffers, these inclusions dissolve, rendering cells practically invisible.

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
Any input, thoughts suggestions and considerations are very welcome!

Thank you for your time!
Artur Zaduryan
Posts: 1
Joined: Feb 07 2018 3:47 am

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