You can stopped the process after adding TRIzol and kept the samples at -80C (even at 20C), but I reccomend freeze the samples straight away.

Hello Ioanna Zarogoulidou

I agree with María Eugenia Cardillo . You can stop the RNA extraction process after adding TRIzol, but the sample has to be frozen immediately and stored at -80°C.

I would not recommend leaving the samples in ice in TRIzol until you get to come back to the lab and then start the RNA extraction protocol. TRIzol's primary function is to immediately stabilize RNA by inactivating RNases and denaturing proteins. Keeping it on ice doesn't improve this primary function and can hinder it. The best approach would be to use commercial RNA stabilizers like RNAlater for sample stabilization at room temperature, which is a convenient alternative to cold storage or immediate TRIzol processing.

Collect the sperm sample, preferably after purification to remove seminal plasma and somatic cells to improve RNA quality. Immediately immerse the sperm pellet in a volume of RNAlater solution. Follow the manufacturer's instructions for the appropriate sample-to-solution ratio. For instance, a 10:1 RNAlater/sample ratio means you should use 10 volumes of RNAlater solution for every 1 volume of your biological sample, ensuring that the sample is completely submerged to maintain RNA integrity and for effective long-term storage. For tissue sample, a 0.5 g tissue sample would require approximately 2.5 mL of RNAlater solution to achieve this ratio. It is recommended that a period of storage at 4°C would allow the solution to penetrate the cells thoroughly before freezing. You may store the semen sample on ice (4°C) for short-term preservation before moving to -20°C or -80°C for long-term preservation.

Please note that to extract RNA from a sperm pellet stored in RNAlater for TRIzol processing, you must first remove the RNAlater by centrifuging the sample. Place the sperm in RNAlater in a centrifuge tube and centrifuge to pellet the sperm. This separates the sperm from the surrounding RNAlater solution. Carefully remove and discard the RNAlater supernatant, leaving the sperm pellet at the bottom of the tube. You can now add TRIzol directly to the sperm pellet to proceed with your standard TRIzol RNA extraction protocol.

Q1. Has anyone stopped the process after adding TRIzol and kept the samples at -80C until further handling?

Yes, that is the correct way of proceeding.

Q2. If so, would it be a problem if I didn't freeze the samples straight away since I still have to keep my samples on ice and transfer them back to the lab a few hours after the TRIzol addition?

It would be a problem. While TRIzol inactivates RNases, RNA can still degrade over time, and a standard ice bath (0–4°C) is not sufficient for proper long-term preservation. To ensure the highest quality and integrity of your RNA, you should proceed with one of these two options immediately after adding TRIzol. You either process the sample immediately according to the protocol or freeze the sample at -80°C for later extraction.

Q3. Would TRItidy be a problem in this case since it doesn’t clearly suggest such pause in the manual?

TRItidy has the same composition as TRIzol and works in a similar manner. So, you can stop RNA extraction process after having added TRItidy, but the sample has to be immediately frozen at -80°C.

Regards,

Malcolm Nobre

Hi Ioanna Zarogoulidou ,

Yes, we can stop the process after adding TRItidy and still keep the RNA intact. Once the sperm cells are lysed in the reagent, the RNases are inactivated, which means the RNA is already protected. It’s a good idea to let the sample sit for a few minutes at room temperature to allow the reagent to fully penetrate and denature proteins. After that, keeping the tubes on ice for a few hours while you transport them back to the lab will not damage the RNA.

If you are not ready to continue immediately, you can place the samples at –80 °C once you reach the lab, and they will remain stable in this condition for weeks to months. The only precaution is to avoid repeated freeze–thaw cycles, so aliquoting is a smart approach if you plan to process them at different times.

Even though TRItidy’s manual doesn’t clearly state this, it functions just like TRIzol since both are phenol–guanidinium based. Many researchers use this pause point without issues. So, in your case, your workflow—collecting sperm, lysing in TRItidy, transporting on ice, and then either continuing straight away or freezing until you’re ready—is absolutely acceptable and should not compromise RNA integrity.

Regards,

Taniya Mary Martin

Thank you all for your answers! This is very helpful information and will really help me distribute my workload much better!

*One more detail: My TRIzol lysing process consists of heating and vigorous vortexing (optimized for the sperm cells) process which takes place after I've transferred the sperm-in-TRIzol samples to the lab. Would it be better for the sperm cells to be fully processed before freezing at -80C or should I perform the additional steps after I've thawed the samples from the -80C freezing?