Dear all,

we have begun using an outsorced laser microdissection system from Carl Zeiss (PALM microbeam) that lifts the microdissected tissue onto adhesive caps of the collection tubes.

For RNA isolation we are using the mirRNAeasy FFPE kit from Qiagen. Our main issue comes from working with these adhesive caps and I would be very grateful for your workflows/suggestions.

We are working on FFPE samples and it was suggested we do an additional deparaffinisation step. As per protocol this has to be done with inverted tubes. Do you have any issues during centrifugation as the fit of the inverted tubes in the centrifuge is not good? How have you fixed this?

The incubation steps for Proteinase K and at 56°C and 80°C for PKD buffer present an issue as the tubes do not fit into the thermomixer. Using a regular hot air incubator has been ruled out by the PI (temperature inaccuracies), as has been the water bath due to fear of leaks.

We have therefore resorted to cutting the tails of the tubes to fit them upside down into the thermomixer. We know we could tape them shut with parafilm, but this introduces a lot of handiwork and processing time for each step and sample.

It has been suggested to use tweezers to chuck the adhesive cap into the tube, but I would like to avoid such manipulation if possible. I am not sure with what these adhesive caps are fitted inside of the tube cap (glue? they get out of the cap fairly easily), and I fear that we could lose the sample/lots of opportunities for contamination when you are trying to loosen the adhesive cap for each and every sample...

What are your experiences with this protocol and how have you worked around these issues?

Best regards,