RNA isolation from Formaldehyde fixed samples

[Pow Joshi]

Dear All,

I have some cells fixed in 4% formalin (neutral I believe). I was wondering
if anyone has tried any RNA extraction on such cell/tissue samples, and if
you have any wisdom including kits, tricks, solutions, problems that you'd
share with me. I would appreciate it hugely.

Thank you much,
Pow

[Pow Joshi]

On 16 April 2013 00:00, DK <d...@no.email.thankstospam.net> wrote:

> In article <mailman.259.136606...@net.bio.net>, Pow Joshi <

> What's the downstream application? How old are fixed samples and
> how were they stored?
>
> I have next to zero experience with what you are facing and I am
> mostly curious as to what the real answer is but my guess is that
> almost all of the RNA (say, 99%) is not recoverable.
>


Well, they have kits for FFPE samples that claim very high recovery. But I
have never used them and didn't know the details except what's written on
the kit. I would love to do some real time pcr on the samples. These cells
were fixed in 2-4% formaldehyde, and kept in the refrigerator 4-8 deg C,
for about 6mo to an yr. I may have some samples that were fixed for 15
min, spun down and re-suspended in PBS for storage. So the formalin
exposure was minimal.
yes, I too believe it may be difficult to recover any RNA from them.
However, I am willing to give it a try if I have some extra information on
the method(s).


> Pow
>
>
>
>
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[Shahrzad Jalali]

Isolation of RNA from formalin fixed paraffin tissue is doable. I have done
it. Use High Pure RNA paraffin Kit from Roche.....

On Tue, Apr 16, 2013 at 12:00 AM, DK <d...@no.email.thankstospam.net> wrote:

> In article <mailman.259.136606...@net.bio.net>, Pow Joshi <
> pow....@gmail.com> wrote:

> What's the downstream application? How old are fixed samples and
> how were they stored?
>
> I have next to zero experience with what you are facing and I am
> mostly curious as to what the real answer is but my guess is that
> almost all of the RNA (say, 99%) is not recoverable.
>

> DK

[Shahrzad Jalali]

Isolation of RNA from formalin fixed paraffin tissue is doable. I have done
it. Use High Pure RNA paraffin Kit from Roche.....


On Tue, Apr 16, 2013 at 12:00 AM, DK <d...@no.email.thankstospam.net> wrote:

> In article <mailman.259.136606...@net.bio.net>, Pow Joshi <
> pow....@gmail.com> wrote:

[Pow Joshi]

On 16 April 2013 13:13, Shahrzad Jalali <shahrza...@gmail.com> wrote:

> Isolation of RNA from formalin fixed paraffin tissue is doable. I have done
> it. Use High Pure RNA paraffin Kit from Roche.....
>

Would you be able to share some of the details and experiences. My cells
are not in paraffin, so I can exclude the Xylene steps. However, I am
concerned about the formaldehyde crosslinks...!

Thanks,
Pow

>
>
> On Tue, Apr 16, 2013 at 12:00 AM, DK <d...@no.email.thankstospam.net> wrote:
>
> > In article <mailman.259.136606...@net.bio.net>, Pow
> Joshi <
> > pow....@gmail.com> wrote:

[Dr Engelbert Buxbaum]

In article <mailman.270.136613...@net.bio.net>,
pow....@gmail.com says...

>
> On 16 April 2013 13:13, Shahrzad Jalali <shahrza...@gmail.com> wrote:
>
> > Isolation of RNA from formalin fixed paraffin tissue is doable. I have done
> > it. Use High Pure RNA paraffin Kit from Roche.....
> >
>
> Would you be able to share some of the details and experiences. My cells
> are not in paraffin, so I can exclude the Xylene steps. However, I am
> concerned about the formaldehyde crosslinks...!

Although methanal (formaldehyde) is a cross-linking fixative, the number
of cross-links introduced per macromolecule is small, especially if the
exposure time is short. FFPE material was usually exposed only for a few
hours to a day, whilst material stored in formalin for years may be more
of a problem.

Techniques that use only a relatively small section of a macromolecule
will still work on FFPE samples. My personal experience is with antibody
binding: polyclonal serums are rarely a problem. With monoclonals the
chance is at least fair, although there are monoclonals that are known
not to work on FFPE sections.

Where I would be more cautious is with quantitative measurements, the
extraction efficiency will invariably be lower than with fresh material,
and may depend on the exact condition that each sample was exposed to
(in particular, exposure time). The solution can sometimes be micro-
dissection where both the experimental and control tissue come from the
same block. In such cases, paired tests may give you valid results.

--
Car (noun): erratically moving obstacle on the road

[Nick Theodorakis]

This paper has some tips:

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001261

In your case, since your samples were stored cold for a year or less, you may be ok. (A bigger problem is retrieval of RNA from archival samples) Be advised that the RNA will likely look degraded by analysis by bioanalyzer or other methods, and even "intact" looking RNA may not amplify well because adducts on the RNA inhibit the progress of polymerases. Keep you amplicons small (which will likely be so if you are doing real time) and, if you prime with oligo-dT, near the 3'end.

Most of the commercial FFPE RNA isolation kits will include an incubation step in Prot. K in slightly alkaline buffer, which are thought to help remove protein cross-linked to the RNA.

Nick

--
Nick Theodorakis

[Nick Theodorakis]

On Tuesday, April 16, 2013 12:48:56 PM UTC-4, Pow Joshi wrote:

[Pow Joshi]

> Nick Theodorakis
>


Thank you, both, Engelbert, and Nick. I will keep these in mind, and look
at the Plosone paper as well. It woudl be awesome if the samples can be
used for real time pcr assays ...

[Pow Joshi]

On 17 April 2013 14:03, Pow Joshi <pow....@gmail.com> wrote:

>
>
> On 17 April 2013 10:54, Nick Theodorakis <nick.the...@gmail.com>wrote:
>

>> Nic
>> Nick Theodorakis
>>
>
>
> Thank you, both, Engelbert, and Nick. I will keep these in mind, and look
> at the Plosone paper as well. It woudl be awesome if the samples can be
> used for real time pcr assays ...
> Pow
>

On a related note, I read the Plosone paper that Nick sent, the proteinase
K and heating to 65-70 deg C is the method to digest the bound proteins and
break the methylene crosslinking bridges. I believe it is done in that
order, protease then heat. I was wondering if one could potentially do it
in the reverse, heat first then add a smaller amount of protease? and would
that lead to a better yield ?.... has anyone tried any such combinations,
and theoretically if there could be problems such as RNA hydrolysis or any
others....

I'd appreciate anything you may have to add to this discussion!

Thank you,

[Shahrzad Jalali]

I had followed exactly the kit direction to isolate RNA from formalin fixed
paraffin embedded tissue. I am not sure how it will work if you use
formalin fixed tissue...you would probably need to give a try.