Re: Re: [Histonet] RNA isolation form stained slides
I read one paper or abstract recently where sections were made at 50
microns then used
successfully for RT-PCR. I am pretty sure that it was PRRS virus that
they were pursuing.
On 3:59, Mark Tarango wrote:
I would try getting more sections from LCM and extracting into 7.5 uL, if
that volume will be enough to for your PCR reactions.
On Friday, November 2, 2012, Vanessa Orsini wrote:
With the kit I'm extracting in 10ulthe problem is that I have too use
few stained cells isolated with the LCM...so even if I increase the number
of slides I'll never have a lot of material...
Inviato da iPhone
Il giorno 2 nov. 2012, alle ore 16:44, Mark Tarango
marktara...@gmail.comjavascript:_e({}, 'cvml',
'marktara...@gmail.com'); ha scritto:
Have you tried using more sections during extraction? Can you extract
into a smaller volume?
On Friday, November 2, 2012, Vanessa Orsini wrote:
Hello,
I need to extract RNA for a RT-PCR after Laser Micro
Dissection on xGal stained slides.
I tried using sections from unfixed frozen organs. I fixed the
sections in EtOH70% for 10min and then I stained them with xGal for 3h at
37°C.
After air drying I cut out with the LCM and extract RNA with the PicoPure
kit
from Applied Biosystem. So far I didn’t manage to get enough RNA.
I tried to add RNase inhibitors to all the solutions but it
didn’t help.
Any idea/suggestion?
Do someone think it would be better to do a LacZ antibody staining
on FFPE sections and extract RNA with an appropriate kit? The RNase would
they
be less active?
Thank in advance for any help you can give me J Vanessa
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Re: [Histonet] RNA isolation form stained slides
Have you tried using more sections during extraction? Can you extract into a smaller volume? On Friday, November 2, 2012, Vanessa Orsini wrote: Hello, I need to extract RNA for a RT-PCR after Laser Micro Dissection on xGal stained slides. I tried using sections from unfixed frozen organs. I fixed the sections in EtOH70% for 10min and then I stained them with xGal for 3h at 37°C. After air drying I cut out with the LCM and extract RNA with the PicoPure kit from Applied Biosystem. So far I didn’t manage to get enough RNA. I tried to add RNase inhibitors to all the solutions but it didn’t help. Any idea/suggestion? Do someone think it would be better to do a LacZ antibody staining on FFPE sections and extract RNA with an appropriate kit? The RNase would they be less active? Thank in advance for any help you can give me J Vanessa ___ Histonet mailing list Histonet@lists.utsouthwestern.edu javascript:; http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RNA isolation form stained slides
I would try getting more sections from LCM and extracting into 7.5 uL, if
that volume will be enough to for your PCR reactions.
On Friday, November 2, 2012, Vanessa Orsini wrote:
With the kit I'm extracting in 10ulthe problem is that I have too use
few stained cells isolated with the LCM...so even if I increase the number
of slides I'll never have a lot of material...
Inviato da iPhone
Il giorno 2 nov. 2012, alle ore 16:44, Mark Tarango
marktara...@gmail.com javascript:_e({}, 'cvml',
'marktara...@gmail.com'); ha scritto:
Have you tried using more sections during extraction? Can you extract
into a smaller volume?
On Friday, November 2, 2012, Vanessa Orsini wrote:
Hello,
I need to extract RNA for a RT-PCR after Laser Micro
Dissection on xGal stained slides.
I tried using sections from unfixed frozen organs. I fixed the
sections in EtOH70% for 10min and then I stained them with xGal for 3h at
37°C.
After air drying I cut out with the LCM and extract RNA with the PicoPure
kit
from Applied Biosystem. So far I didn’t manage to get enough RNA.
I tried to add RNase inhibitors to all the solutions but it
didn’t help.
Any idea/suggestion?
Do someone think it would be better to do a LacZ antibody staining
on FFPE sections and extract RNA with an appropriate kit? The RNase would
they
be less active?
Thank in advance for any help you can give me J Vanessa
___
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RE: [Histonet] RNA isolation form stained slides
Have you tried to extract the RNA from the tissue without the stain- just to test your method? I am not familiar with the mechanics of the stain but you might be denaturing the RNA. Also are you sure there is enough message in the area you are trying to extract to get enough RNA? Maybe you need more material. Sue Hunter, Supervisor Advanced Diagnostics Beaumont Health System Royal Oak MI 248-898-5146 shun...@beaumont.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Vanessa Orsini Sent: Friday, November 02, 2012 5:43 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] RNA isolation form stained slides Hello, I need to extract RNA for a RT-PCR after Laser MicroDissection on xGal stained slides. I tried using sections from unfixed frozen organs. I fixed the sections in EtOH70% for 10min and then I stained them with xGal for 3h at 37°C. After air drying I cut out with the LCM and extract RNA with the PicoPure kit from Applied Biosystem. So far I didn't manage to get enough RNA. I tried to add RNase inhibitors to all the solutions but it didn't help. Any idea/suggestion? Do someone think it would be better to do a LacZ antibody staining on FFPE sections and extract RNA with an appropriate kit? The RNase would they be less active? Thank in advance for any help you can give me J Vanessa ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
