RNA and intracellular labelling
Sian.Rizzo at icr.ac.uk
Wed Nov 30 05:10:25 EST 2005
there is a clear need for a concerted effort to get good quality RNA
from cells following intracellular antibody labelling.
Although I've not used cells from sorting, our group has tried to get
around similar problems and so you might find our endeavours of value.
We (mostly Jenny Knight, for her PhD) are trying to do laser-capture
microdissection from frozen tissue sections following IF.
What we know so far:
Tissue sections are cut as and when needed as once cut then refrozen
(not fixed) the RNA degrades slowly, so no good after a month at -80.
Many antibodies/general reagents may have RNAases or chemicals that
degrade RNA - use RNA inhibitors (like the ones used in reverse
transcription), buckets if using antibodies in supernatants
RNAase-free everything inc.all buffers (so if cells are dehydrated in
alcohols, fine, but the minute they are back in aqueous solutions
degradation begins EVEN though fixed!!) & use virgin plastics ie. we use
sterile 50ml tubes for washing/dehydration steps, make up fresh
complete dehydration reduces degradation, we are lucky in that we have
to dehydrate through alcohols to xylene before laser capture giving us a
breather in the race to collect cells
speed essential - minimise antibody incubations/increase antibody concs
where possible (Jenny managed to reduce a 2hour protocol for indirect
labelling + counterstain to 20mins)
In spite of all that, you are highly unlikely to get top quality RNA
(ie. first the ratio of 18/28S peaks declines and then disappear
altogether, average size of mRNA slides ever lower down your gel). We've
been using picochips + bioanalyser to monitor our RNA quality.
Even with what appears to be reasonable RNA, mostly it doesnt work well
for RT-PCR, as its too fragmented (eg. for 150-250bp amplification)
Forget about polyA tails, use random primers
However, if we amplify the RNA (can do 2 rounds) and use for
microarray, we've found that we can reliably pick up high expression of
the mRNA of the protein that we've used to select cells (Hurrah!)
eg. we used keratin 14 antibody vs. neg cells and found a 5-20-fold
enrichment of mRNA in selected pops, this was supported by a whole bunch
of other genes we expected to see differentially expressed.
This information will be presented at the British Prostate Group Autumn
(!?) Meeting on Monday by Jenny Knight
I hope this helps, i'd love to hear about other experiences, as I am
interested in looking at RNA post-sorting, particularly if anyone has
tried allowing an in vitro recovery time following sorting to see if the
RNA profile is altered/restored etc.
Prostate Stem Cell Laboratory
Male Urological Cancer Research Centre
Institute of Cancer Research
15 Cotswold Road
Surrey SM2 5NG
TEL: +44(0)20 8722 4178
FAX: +44(0)20 8722 4278
email: Sian.Rizzo at icr.ac.uk
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