Hello, I'm planning to sample live snails and ship them internationally.
My goal is to immerse live snails in liquid nitrogen to reduce expression noise, then grind the tissues and use a combination of RNAlater and cold storage for about a couple of months before internationally shipping them to another country for mRNA extraction (takes about a week).
The problem I have as of now is that ThermoFisher manual states that RNAlater is not suitable for previously frozen samples. They recommend RNAlater-Ice as substitute but RNAlater-Ice only lasts about 30 minute at room temperatures. Dry-ice shipment is also out of my budget.
I was wondering if you have any solutions to this dilemma, how can I preserve the RNA integrity best in this scenario?
Would it be possible to transition from RNAlater-Ice to RNAlater?
Thank you in advance!
Transitioning from flash freezing snails to using RNAlater for long-term storage can help preserve RNA integrity. Here's a step-by-step guide to ensure a successful transition without sacrificing RNA quality:
1. Prepare the snail samples: Collect the snail samples and handle them carefully to minimize RNA degradation. Keep the samples on ice throughout the process to maintain their integrity.
2. Flash freezing: If you have been using flash freezing for snail storage, continue with this step. Place the snail samples in liquid nitrogen or a dry ice-ethanol bath for rapid freezing. This method quickly immobilizes cellular processes, minimizing RNA degradation.
3. Transfer to RNAlater: Once the snails are flash frozen, you'll transition to RNAlater for long-term storage. RNAlater is a solution designed to stabilize RNA in tissues and cells. Thaw the samples slightly by placing them on ice, but do not let them fully thaw.
4. Add RNAlater: Carefully transfer the flash-frozen snail samples into microcentrifuge tubes containing RNAlater. Ensure that the samples are fully submerged in RNAlater to maximize RNA preservation. Use a sufficient volume of RNAlater to cover the snails completely.
5. Store the samples: Seal the microcentrifuge tubes containing the snail samples in RNAlater and store them at an appropriate temperature. The recommended storage temperature for RNAlater is usually between 2-8°C (refrigerator temperature). Check the specific instructions provided by the manufacturer of the RNAlater solution.
6. Long-term storage: RNAlater is designed for long-term storage of RNA. It helps to prevent RNA degradation by creating a protective environment that stabilizes the RNA molecules. Keep the samples in RNAlater for the desired storage period, depending on your experimental needs.
7. RNA extraction: When you are ready to extract RNA from the snail samples, remove them from RNAlater and proceed with the RNA extraction protocol of your choice. RNAlater is compatible with most RNA extraction methods, and the samples can be directly used for downstream RNA analysis.
It's important to note that while RNAlater is effective in preserving RNA integrity, it's still crucial to handle the snail samples with care during the transition process. Minimize exposure to RNases (ribonucleases) by using clean, RNase-free equipment and working in a dedicated RNA-friendly workspace.
Always follow the specific instructions provided by the manufacturer of the RNAlater solution to ensure optimal storage conditions and RNA preservation. Additionally, it's advisable to perform quality control checks, such as assessing RNA integrity using methods like gel electrophoresis or RNA quantification techniques, to confirm the integrity of the extracted RNA after storage in RNAlater.
Hi Gopal, thanks for the detailed guide. I have decided to just use RNAlater-ICE to transition from flash freezing.
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