Re: [Histonet] OCT embedding...
We have recently aquired a laser capture microdissection system and have
been embedding our tissue in OCT, capturing tissue for RT-PCR without much
problem (although we are still quite new to this!). We just make sure that
all our instruments that we use to take, embed and section the biopsy has
been sterilized and washed in RNAse Away. Also we make sure that we embed
as quickly as possible and store our blocs at -80deg. Our tissue is
sectioned at -35 (we are working with skin) and sections are kept cold
(store in -80) until use (we clean the cryostat out inbetween each bloc).
We have an extremely fast staining protocol (I was surprised by how little
time immuno could take!) that we adapted from Lindeman N et al.
(Diagnostic Molecular pathology 2002 11(4):187-192) and we use a kit
(picopure) for our RNA. So far we have had good results and it didn't take
much time to set the procedures up. We have had good technical assistence
from the LCM manufaturer (Arcturus) though. Check out the Arcturus web
site if you need more technical info.
> Dear All,
> I am currently using OCT as an embedding medium for fresh frozen tissue.
> Ideally i need to cut sections from this embedded tissue, laser dissect
> specific areas of interest and extract high quality RNA from the lasered
> tissue for subsequent RT-PCR and microarray analysis. Does anyone know
> if OCT medium degrades RNA?...if so can RNase inhibitors be added to
> such solutions?
> Also does the actual embedding procedure itself comprise RNA
> integrity?...does anyone have a good protocol for this techniwque and
> subsequent good quality RNA extraction?
> Rebecca Hands (MSc)
> PhD Research Fellow
> Academic Dept of Surgery
> 4th Floor Alexandra Wing
> The Royal London Hospital
> Whitechapel Road
> E1 1BB
> Ext 2614
> Histonet mailing list
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