Extract RNA from Sockeye Samples

[sr320]

Samples in the bottom of -80.

Want to extract 4 samples from each tissue to determine quality and quantity.

Prior to extracting lets confirm plan.. For example do we split samples if large tissue mass.

[kubu4]

For posterity, copying Slack convo to here...

we can isolate RNA from most everything. Depends on if I should try to save some tissue for other things or use it all for RNA. Also, not sure on expected RNA yields for all of the different tissue types. Also, a fair number of blood collected in RNAlater have cracked (they were overfilled before being frozen) and they will be particularly difficult to handle easily for RNA isolations:

I'm also not entirely sure how to handle the blood stored directly in the Vacutainers. There's a lot of blood in each container. Assuming there's a fair amount of cell lysis due to freezing, which could make it difficult to (easily) get RNA. Plus, would have to thaw entire container to allow mixing before sampling. Less than ideal, but if kept cold while thawing, would probably work.

Also, will this be for RNAseq and/or qPCR? Wouldn't need much RNA for qPCR, but would obviously need more for just RNAseq.

Would like to use TriReagent with Direct-zol kit from Zymo ($250). Can I order using money from Jackie's supply reimbursement?

[sr320]

Lets plan on extracting 4 brain, liver and gonad samples. (do nothing with blood)

Does size of brain, liver and gonad samples suggest extracting whole samples or splitting?

[kubu4]

Lets plan on extracting 4 brain, liver and gonad samples

Does this look like this:

2 x brain Territorial 2 x brain Social

2 x gonad Territorial 2 x gonad Social

2 x liver Territorial 2 x liver Social

Does size of brain, liver and gonad samples suggest extracting whole samples or splitting?

This is the big question. Are these being used for RNAseq or qPCR? Liver and gonad can probably be split, as I feel like these two tissues generally have high yields of RNA. I haven't previously done anything with brain, so not sure on yields. Also, in regards to the brain tissue, I think you had said you had some concerns regarding sampling consistency across all samples (e.g. some samples brain stem was collected, other samples no brain stem; some had partial brain, some had whole brain, etc). So, it might be better to use the entirety of the brain samples to minimize tissue heterogeneity between samples?

[sr320]

Likely RNA-seq and qPCR in future. Let proceed how you have suggested.

[kubu4]

Isolated RNA from four individuals (2 in each "treatment" group):

Sample	Tissue	Concentration (ng/uL)	Yield (ng)
2A	brain	0	0
2B	liver	190	9500
2C	gonad	196	9800
3A	brain	0.602	30.1
3B	liver	186	9300
3C	gonad	51.8	2590
23A	brain	0	0
23B	liver	200	10000
23C	gonad	200	20000
25A	brain	20.8	1040
25B	liver	200	20000
25C	gonad	114	22800

The biggest thing here is the poor results from the brain tissue. The other kicker is that I used the entirety of the brain for each of these individuals (to minimize heterogeneity across individuals that might be caused by inconsistent brain tissue collection and prevent introduction of heterogeneity if I were to attempt to dissect a portion of each brain tissue), so those brain samples are gone. Ugh.

I have some ideas on how isolation could be improved, so was thinking I might attempt isolations from another set of tissue sets in order to have a full "suite" of RNA from any given individual's tissue set.

What do you think?

[sr320]

You mean do a couple of additional brains in order to see if yield could be improved?

[kubu4]

Yes, and the corresponding liver/gonad for those other individuals. Wasn't sure how this project was set up, so was thinking gene expression wanted to be compared between tissues within an individual.

[sr320]

It is more - within tissue- so just two more brains would be fine