What analyses should I have performed on this tissue?

[Meredith L. Patterson]

(PSA: contains frank and clinical discussion of suicide, suicidality.)

So, as you probably all know, my husband, who was also a DIYbio-er, killed himself in 2011. I asked the autopsy director to collect and cryopreserve a variety of tissue and brain structure samples (hypothalamus, pituitary gland, adrenal glands, raphe nuclei, locus coeruleus, amygdalae, hippocampus, peripheral nerve tissue related to previous EMG results -- I haven't actually laid eyes on the samples yet). I've been waiting for sequencing costs to come down, and all of a sudden it turns out I have a colleague in China with contacts in biotech at East China Normal University, so he's looking into what kinds of sequencing operations they can perform.

It turns out ECNU does a giant crapton of brain functional genomics: http://sbg.ecnu.edu.cn/na_en/research_labor.html

I'd kinda put this on the "yeah, it'll happen someday" list but it's increasingly looking like "someday" is now. Any brain functional genomics folks have advice on where to start while I refresh myself on my notes from three years ago?

Cheers,

--mlp

[SC]

Do you have a particular question you're trying to answer?

 

Also, keep in mind that a lot of functional genomics involves the analysis of RNA, not DNA.  I'm not sure intact RNA can be retrived from a cadaver.

[Steve]

On Monday, 8 December 2014 18:01:33 UTC, SC wrote:

".....I'm not sure intact RNA can be retrived from a cadaver."

on that point, any particular reason why not?  at approximately 10 picogram per mammalian cell, there is certainly enough of it.  (And given that probing for viral RNA is used in screening donor organ tissue, which would be at much lower overall concentrations in cell lysates).
Certainly at -80, useful data can be retrieved from tissue or lysates after years.

[Cathal Garvey]

I suspect the difference is that organs for donation are still "viable";
live cells, active cytoplasm, ongoing RNA turnover and transcription,
however slow. After death without preservation, I think most relevant
tissues, particularly in the brain, would suffer cell death pretty
quickly, with RNA degradation being one of the fastest processes in that
chain of events.

Not being an expert on RNA metabolism or isolation though, I'm guessing
here.

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[Bryan Bishop]

On Mon, Dec 8, 2014 at 10:21 AM, Meredith L. Patterson <clon...@gmail.com> wrote:

I'd kinda put this on the "yeah, it'll happen someday" list but it's increasingly looking like "someday" is now. Any brain functional genomics folks have advice on where to start while I refresh myself on my notes from three years ago?

Just off the top of my head, and not a thorough list:

* Whole genome sequencing, and other variations of sequencing

* mRNA/transcripts, although the number of required samplings would make me think this could only get done as a DIY project (I don't see anyone doing neural tissue transcriptomics on scienceexchange)

* High-resolution scans, http://3scan.com/

* One day in the near future: synapse-resolution scans, http://brainbackups.com/

* Generally, there are many antibody-based tagging/visualization systems for fixed tissue. You'll have to browse through some antibody catalogs to figure out which particular methods would be most interesting to you. There are many antibodies or aptamers that can be made to specifically extract certain details from these samples.

- Bryan
http://heybryan.org/
1 512 203 0507

[Josiah Zayner]

One would assume that with any tissue harvesting the sample would either be fixed or cryopreserved meaning the RNA should still be just fine.

[Jeswin]

On Sun, Dec 14, 2014 at 4:20 PM, Josiah Zayner <josiah...@gmail.com> wrote:
> One would assume that with any tissue harvesting the sample would either be
> fixed or cryopreserved meaning the RNA should still be just fine.
>

I'm no expert, but from the extractions I have done of mammalian RNA
from FFPE slices or tissue stored in RNAlater, you will run into
degraded RNA. Degradation is usually caused by RNases, especially if
the samples are not preserved as soon as possible. Since RNases are
some common and hard to get rid of, proper preservation techniques are
the key.

That said, degradation may not have much affect on sequencing. My
colleagues have sequenced with degraded RNA but I think intact RNA
(RNA Integrity Number > 8) is preferred. I will have to talk to them
to find out.