[Histonet] Rnase free slides?
amosbrooks <@t> gmail.com
Wed Jul 7 19:40:17 CDT 2010
As you said RNAases are everywhere, so chasing them all down would be
akin to hunting the boogyman. The way I see it is since there is no
realistic way of eliminating any contact at all with RNAase it really comes
down to merely damage control. Start with gloves, you reduce the
introduction of RNAase by a certain percentage. Wipe down all the surfaces
that come in contact with the tissue with RNAase away, you reduced the
percentage a bit further. Only use DEPC treated water and you reduce the
percentage further and so on until it gets to be so labor intensive that it
is non productive. You can hold your breath to reduce them too... don't
laugh I found myself doing it unwittingly one day.
So what happens if we scale it down to a workable level? Some RNAases
will inevitably be introduced this will reduce the PCR yield some. If you
are doing it a certain way and your yields are sufficient, it aint broke so
don't fix it! So if you are using untreated slides and it's working, don't
change a thing. That having been said I never assume anything is RNAase
free. This includes the slides and any chemicals I use, previously opened or
not. So if it doesn't take much effort to RNAase away treat something I
usually do it. Otherwise I just chalk it up to acceptable yield loss.
Incidentally I meant 30% sucrose when I said 30% in my last post. I've
never done a side by side comparison of the difference between 30% sucrose
in DEPC and OCT. There may be no appreciable difference at all. I merely
mentioned it as an option.
So a funny story: I had a researcher really giving me H-E-double hockey
sticks wanting me to make absolutly sure there was no possibility of RNAase
contamination. (Thus the holding my breath I mentioned earlier.) So I went
into full OCD mode and decontaminated everything in RNAase away including
the box I put the slides in. The researcher picked up the slides and after
grilling me again about my technique he popped open the box (bare-handed,
mind you) to check the slides out and ran his finger down the sides of the
slides. I literally saw the RNAase bugs eating away all my hard work before
my eyes. When I caught my breath I took a long lunch and went home early.
Later that week I saw the researcher. He said the yield was great and I must
have followed his advise well. That was a Scotch night! RNAase ... the
boogeyman and Big Foot!
On Wed, Jul 7, 2010 at 7:32 PM, Emily Sours <talulahgosh <@t> gmail.com> wrote:
> Am I wrong in assuming that the glass slides I get (which are in a
> plastic box, wrapped in plastic) are not RNase free? They're
> superfrost plus from Fisher. It hasn't been a problem for us to
> assume so, but I'm wondering what other labs think about it.
> We've never treated our slides with Rnase Away or rinsed in DEPC
> water. We use DEPC water for in situ hybridization solutions. The in
> situ dishes are sprayed with RNase away, then DEPC water.
> Also use OCT when you section, it'll make your life so much easier!
> It's assumed in my lab that if a chemical came in and hasn't been
> opened, it's RNase free. To keep it RNase free, you always wear
> gloves. Store your slides in a new box to keep them RNase free.
> RNases are everywhere on people, I assumed, and that was the problem.
> I didn't think RNases are everywhere in the universe on every object.
> But I could be wrong. Anyone else have an opinion?
> Dark Pictures, thrones and stones that pilgrims kiss
> And poems that take a thousand years to die
> But ape the immortality of this
> Red label on a little butterfly.
> -Vladimir Nabokov, concluding stanza of ‘A Discovery’ 1941.
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