Hi,

Your RNA is not completely degraded within this one minute. However, you should keep the time outside of the -80 as short as possible. If you can avoid thawing your samples prior to RNA extraction, it would be better.

Complete degradation of RNA never takes place within a short time. Take plant material from -80 and immediately isolate the RNA, that will help to yield better RNA concentration.

Thank you both....

Like the others said, I think you are okay...at the very worst you may get some reduction in yield, but i think your good.

I have never worked with plant material, only animal tissue. One of the things I do is place the tissue tubes from the -80 directly on a bed of dry ice in a large rectangular pan. I then cut my tissue in a small weigh pan directly on the dry ice. I then weigh the sample (if needed) moving it with cold forceps. The idea is to keep the sample frozen and cold as long as possible prior to disruption. Everything stays in/on the dry ice until I load my lysis buffer and put my samples into the tissue-lyser.

I was also told that it is the number of freeze-thaw cycles that greatly degrades RNA (along with letting it get warm) in a sample. I don't know if you are taking samples from a larger section/piece but the idea is to reduce or not have a number of freeze-thaw cycles in your sample and or your stock material. I hope this helps.

I will only take my -80C sample out for RNA extraction when all the tools (bowl and pestle) are ready. I will pour some liquid N2 inside the bowl with pestle to make them as cold as possible, then take out the sample from -80C and directly into the bowl for grinding.

Also, take a look at the paper below: "Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue." They have found that Minimal RNA degradation was detected after 30 minutes of thawing in unfixed samples. In its conclusion: degradation of RNA in frozen tissue occurs first after several minutes of thawing. Already minimal decrease in RNA quality may result in significant changes in gene expression patterns in clinical tissue samples.

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Diagn Mol Pathol. 2009 Mar;18(1):44-52. doi: 10.1097/PDM.0b013e3181857e92.

Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue.

Botling J1, Edlund K, Segersten U, Tahmasebpoor S, Engström M, Sundström M, Malmström PU, Micke P.

Author information

1Department of Genetics and Pathology, University Hospital, Uppsala, Sweden.

Abstract

Biobanks of fresh, unfixed human tissue represent a valuable source for gene expression analysis in translational research and molecular pathology. The aim of this study was to evaluate the impact of thawing on RNA integrity and gene expression in fresh frozen tissue specimens. Portions of snap frozen tonsil tissue, unfixed or immersed in RNAlater, were thawed at room temperature for 0 minute, 5 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 16 hours before RNA extraction. Additionally, tonsil tissue underwent repetitive freezing and thawing cycles. RNA integrity was analyzed by microchip gel electrophoresis and gene expression by quantitative real-time polymerase chain reaction for selected genes (FOS, TGFB1, HIF1A, BCL2, and PCNA). Minimal RNA degradation was detected after 30 minutes of thawing in unfixed samples. This degradation was accompanied by relevant changes in gene expression for FOS and BCL2 at 45 minutes. Modified primer design or the use of different housekeeping genes could not rectify the changes for FOS. Repetitive thawing cycles had similar effects on RNA integrity. The incubation of the tissue in RNAlater efficiently prevented RNA degradation. In conclusion, degradation of RNA in frozen tissue occurs first after several minutes of thawing. Already minimal decrease in RNA quality may result in significant changes in gene expression patterns in clinical tissue samples.

Yuan-Yeu Yau Thank you for detailed answer..