UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.
The RNA isolation I performed earlier this week proved to be better for some of the samples (scraping tissue directly from the blocks), but still exhibited low yields from some samples. I will perform a final RNA isolation attempt (the kit only has six columns left) from the following samples:
Instead of full sections from each histology cassette, I gouged samples directly from the tissue in each of the blocks to maximize the amount of tissue input.
- Used Buffer TR1 + β-mercaptoethanol (β-ME) prepared 201500505 as well as fresh Buffer TR1 + β-ME prepared today.
- I used aliquots of DNase prepared on 20150408.
Samples were then processed with the PAXgene Tissue RNA Kit in a single group.
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.
All samples were stored @ -80C in Shellfish RNA Box #5.
Two samples (02 and 07) produced great yields and perfect RNA (260/280 and 260/230 of ~2.0). The remainder of the samples showed little improvement compared to what I’ve been obtaining from the previous three attempts. Will discuss with Steven and Brent about how to proceed with this project.