RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

Last week’s RNA isolation failed for more than half of the samples I processed. I will re-isolate RNA from the following samples:

IMPORTANT:

I prepared fresh Buffer TR1 + β-mercaptoethanol (β-ME).
I used aliquots of DNase prepared on 20150408.

Five 5μm sections were taken from each block. A new blade was used for each block.

Samples were then processed with the PAXgene Tissue RNA Kit in two groups of six.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

“Max speed” spins were performed at 19,000g.
Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.
Shaking incubation step was performed with Disruptor Genie
Samples were eluted with 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Results:

(http://eagle.fish.washington.edu/Arabidopsis/20150427_geoduck_gonad_DNased_RNA_ODs.JPG)

(http://eagle.fish.washington.edu/Arabidopsis/20150427_geoduck_gonad_DNased_RNA_plots.JPG)

Well, these results are very consistent with the data from the last isolation performed on these samples. This fact suggests that the problem lies with the tissue samples and not the isolation (since the isolation has been performed two separate times on these same samples and the results have come out virtually identical both times).

All samples with concentrations < 5ng/μL were discarded. The remaining samples were stored @ -80C in Shellfish RNA Box #5:

Will discuss with Steven, look at Grace’s notebook to review the preservation process for these samples, and review the PAXgene Tissue RNA Kit to see if it will accommodate a greater number of microtome sections to use for isolation.