My previous go at this was a little premature – I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that [Grace has gone through and annotated the blocks to point out gonad tissue](RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks).
Isolated RNA from Olympia oyster gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Laura’s notebook for full details on samples and preservation.
RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.
Background on all of this is in this GitHub Issue
- Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1)
Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:
- “Max speed” spins were performed at 20,000g.
- Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
- Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice.
Samples were not quantified due to lack of proper RNA Qubit assay AND the computer that our NanoDrop1000 is hooked up to is dead. Will have Katherine Silliman perform quantification.
Samples were stored at -80C temporarily.
Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.
from Sam’s Notebook http://onsnetwork.org/kubu4/2017/07/19/rna-isolation-olympia-oyster-gonad-tissue-in-paraffin-histology-blocks-2/