Hello!
We want to isolate RNA from mosquitoes that have been infected with a virus in the BLS3 lab using Trizol.
Unfortunately, we cannot perform the Trizol purification in the lab - but we also cannot transfer the mosquito bodies unless the entire tissue is completely diffused with trizol (thus inactivating the virus).
Does anyone have experience with the time required for the complete diffusion of the sample? I realise that it is dependent on the makeup of the sample - but maybe there are papers out there that have compared different samples (like mouse organs or so), which we can then use as a guideline.
Thanks in advance!
I have used TRI reagent for extraction of HCV RNA from Peripheral blood Mononuclear cells (PBMCs). HCV RNA has been successfully isolated and purified. The concentration has been measured. You know that RNA is very sensitive to temperature so always use ice when you transport or thawing the sample.
Hi Mayke,
We often collect mosquitoes which mayou be DENV infected and don't have the possibility to extract RNA immediately. If you use Trizol the best solution is to add between 500ul and 1 ml of Trizol (I usually add 700 ul) to the mosquito(es). Then grind them very well for 2/3 minutes using a pestel. You can use an electric pestel but be careful not to heat too much the sample.
At this point you can wait for other 2/3 minutes and store your grinded mosquito + trizol at -80.
If you need to transfer the samples use dry ice and a thermal bag.
For the extraction let the defreeze and process the samples as usual.
Another very good solution is to use a buffer such as DNA/RNA shield by zymo tech. You can grind mosquitoes in 400 ul of DNA/RNA shield and then just store them at 4°C or -20 for as long as you want or even at RT if you will use themy in few days.
These buffer usually inactivate all viruses and RNases, so they are very good to be used with Aedes.
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