UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.
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 the following samples using the PAXgene Tissue RNA Kit (Qiagen) from the following geoduck sample blocks:
- I used Buffer TR1 + β-mercaptoethanol (β-ME) prepared on 20150408 for samples 02, 03, 04, 07. The rest of the samples were processed with buffer prepared today.
- 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 eight.
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.
Well, these results are certainly not good.
The first set of eight samples I processed yielded no RNA (except #38, which is only marginally better than nothing). All the samples (excluding #38) have been discarded.
The second set of eight samples I processed range from amazing to poor (#68 was barely worth keeping).
I’ll review the protocol, but at the moment I’m at a loss to explain why the first set of eight samples came up empty. Will perform another on these blocks on Monday. Grrrrr.
Samples were stored at -80C in Shellfish RNA Box #5.