I recently sent some RNA lyophilized samples to a sequencing facility. As the samples were lyophilized, I can sent them at room temperature and I did not expect any degradation. However, they did not pass the quality control: RNA was degraded or the amount was not enough, according the Bioanalyzer. I sent 3000 ng of RNA (according to Nanodrop) when the required material is 1000 ng and in some samples the Bioanalyzer quantification was lower than 10 ngr.

We analyzed the quality of the RNA samples after the extraction (before lyophilization) by an electrophoresis agarose gel and with Nanodrop. All samples showed a correct pattern in the gel (strong band of rRNA and no degradation) and proper 260/280 ratios and RNA concentrations in the Nanodrop (all samples had more than 4000 ng). All samples were resuspended in DEPC water.

The day of the sample shipment, a recipient from the equipment, a bottle with an adapter to connect to the equipment was placed at -80 C and when the equipment was ready, the samples and the recipient were taken out of the freezer. The eppendorf tubes were opened and put carefully inside the recipient that was connected to the vacuum pump of the equipment. The equipment was in room at 20-22 C. The process started and I was checking the tubes all the time. After 15 min, I could no longer see any liquid in the tubes, so I disconnect the equipment and I prepare the sample shipment.

I was wonder if the degradation was the lyophilization as samples were 15 min at room temperature until they were lyophilized; nevertheless it is complicate that RNAses act during this period as the vacuum pump creates a negative pressure environment. In the lab, we made a test with other RNA samples and the lyophilizer, and we did not see differences in the Nanodrop measures before and after lyophilization protocol. Besides, I think that RNAses can't act once the samples are lyophilized.

I do not know if anybody has experience on the procedure and could help us?

Thanks a lot for your help

Jose