I’ve been asked to isolate RNA from some paraffin-embedded Olympia oyster gonad tissue.
Despite some excellent documentation by Laura Spencer (
images of tissue layouts in histology cassettes and a corresponding cassette mapping key file), the histology facility seems to have flipped some things around and/or repositioned/split the contents of each cassette. This makes ID-ing the proper tissues tedious and, at times, difficult.
The list of tissues that needs to be processed is listed in this
GitHub Issue #648. I’ve also added the list below:
Prior to beginning RNA isolations, I have annotated images of the histology blocks and will be waiting for Laura to confirm that my annotations are correct. I will be posting a link to this notebook entry in the GitHub issue listed above for her to view and wait for her confirmation.
UPDATE 201700707 – Laura has indicated that many of my annotations are incorrect. Katie has gone through and made proper identification:
Additionally, as indicated in the GitHub Issue above,
histology block “Oly 14″ does not have a corresponding tissue cassette photo (containing sample NF-10 26). Without the original image, I don’t think I can make an accurate guess on how the tissues are oriented in the resulting two histo blocks (see below).
BLOCKS 14 (unable to annotate at time of posting)
Miscellaneous and tagged gonad, histology, histology blocks, histology slides, Laura Spencer, NF-10 22, NF-10-23, NF-10-24, NF-10-26, NF-10-28, NF-10-30, olympia oyster, Ostrea lurida, pics, SN-10-16, SN-10-17, SN-10-20, SN-10-25, SN-10-26, SN-10-31 on . July 5, 2017 Leave a comment
Received histology blocks and slides for Laura Spencer.
Samples were stored in glass cabinet in FTR 213 (see images below).
Isolated RNA from geoduck gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks.
See Grace’s notebook for full details on samples and preservation.
RNA was isolated from only two samples using the PAXgene Tissue RNA Kit (Qiagen) from the following geoduck sample blocks to test out the kit:
Prior to beginning, I prepared Buffer TR1 by adding 10μL of β-mercaptoethanol (β-ME) to 1000μL of Buffer TR1). This will be good for up to six weeks at RT.
Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the “-20C Kit Components” box.
Five 5μm sections were taken from each block.
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 34μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.
Samples were stored at -80C in
Shellfish RNA Box #5.
NOTE: The spreadsheet linked indicates other samples exist in the slots that I placed these two samples. Will need to update the spreadsheet to be accurate.
Looks like the kit worked! Yields are pretty good (~800ng) from each. The 260/280 ratios are great for both samples. Oddly, the 260/230 ratios for the two samples are pretty much polar opposites of each other; not sure why.
Will proceed with the remainder of the samples that were selected by Steven and Brent. Or, maybe I should try to make some cDNA from these RNA samples to verify the integrity of the RNA…
Protein expression profiles during sexual maturation in Geoduck and tagged Disruptor Genie, geoduck, gonad, histology, histology cassettes, NanoDrop1000, Panopea generosa, paraffin, PAXgene Tissue RNA Kit, RNA isolation, RNA quantification on . April 8, 2015 3 Comments