UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.
Last week’s RNA isolation (a second attempt at obtaining RNA from the samples) performed poorly. I will re-isolate RNA from the following samples:
Instead of full sections from each histology cassette, I gouged/shaved off samples directly from the tissue in each of the blocks to maximize the amount of tissue input. However, due to the small size and susceptibility to flying around because of static electricity, none of these were able to be weighed prior to processing.
- Used Buffer TR1 + β-mercaptoethanol (β-ME) prepared 20150427 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.
Well, despite the low numbers, all of the samples (excluding 46 – 68) are double the yield of what I saw previously. This is good, but the amount of RNA from these is probably borderline sufficient quantity for RNA-Seq.
The kit has enough columns for six sample preps. I think I’ll attempt this strategy again (gouging/shaving directly from tissue in histo cassette), but really take a fair amount of tissue this time and see if I can get more.
All samples were stored @ -80C in Shellfish RNA Box #5.